Plant sorbitol biosynthetic

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a sorbitol biosynthetic enzyme. The invention also relates to the construction of a chimeric gene encoding all or a portion of the sorbitol biosynthetic enzyme, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the sorbitol biosynthetic enzyme in a transformed host cell.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/092,952, filed Jul. 15, 1998.

FIELD OF THE INVENTION

[0002] This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingsorbitol biosynthetic enzymes in plants and seeds.

BACKGROUND OF THE INVENTION

[0003] Sorbitol (D-glucitol) is an acyclic polyol found in a number ofplant species (Kuo et al. (1990) Plant Physiol 93:1514-1520). Sorbitolis the primary photosynthetic product in rosaceous fruits and canaccount for a major portion of the carbon transported from the leaf. Incorn sorbitol is found in seed and silk but not in pollen and leaf andlow amounts of sorbitol are detectable in developing corn kernels.Sorbitol is found in soybeans and it is suggested that the accumulationof sorbitol may play a role in facilitating hexose metabolism ingerminating seedlings (Kuo et al. (1990) Plant Physiol 93:1514-1520).During germination, soybeans convert oil and soluble oligosaccharidesinto sucrose which is in turn converted to glucose and fructose to fuelrapid growth. Some investigators have speculated that since plantfructokinases exhibit strong substrate inhibition by fructose, thepresence of a sorbitol pathway may provide a mechanism to bypass thisinhibition by converting excess fructose into sorbitol. This would helpfacilitate the metabolism of free glucose and fructose.

[0004] The metabolism of sorbitol has been extensively studied inseveral plant species (Kuo et al. (1990) Plant Physiol 93:1514-1520,Loescher (1987) Physiol. Plantarum 70:553-557, Loescher et al. in:Photoassimilate Distribution in Plants and Crops, ed. Zamski et al.Marcel Dekker, Inc., New York). There are several enzyme activitiesinvolved in sorbitol metabolism. Three of these enzymes are aldehydereductase (NADPH-dependent aldose 6-phosphate reductase), sorbitoldehydrogenase, and NADP-dependent D-sorbitol-6-phosphate dehydrogenase.Aldehyde reductase appears responsible for the conversion of glucose tosorbitol; NADP-dependent D-sorbitol-6-phosphate dehydrogenase is alsoinvolved in sorbitol synthesis, and sorbitol dehydrogenase is involvedin the conversion of sorbitol to fructose. Accordingly, the availabilityof nucleic acid sequences encoding all or a portion of these enzymeswould facilitate studies to better understand carbohydrate metabolismand function in plants, provide genetic tools for the manipulation ofthe sorbitol biosynthetic pathway, and provide a means to control carbonpartitioning in plant cells.

SUMMARY OF THE INVENTION

[0005] The instant invention relates to isolated nucleic acid fragmentsencoding sorbitol biosynthetic enzymes. Specifically, this inventionconcerns an isolated nucleic acid fragment encoding an aldehydereductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase orsorbitol dehydrogenase and an isolated nucleic acid fragment that issubstantially similar to an isolated nucleic acid fragment encoding aaldehyde reductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenaseor sorbitol dehydrogenase. In addition, this invention relates to anucleic acid fragment that is complementary to the nucleic acid fragmentencoding aldehyde reductase, NADP-dependent D-sorbitol-6-phosphatedehydrogenase or sorbitol dehydrogenase.

[0006] An additional embodiment of the instant invention pertains to apolypeptide encoding all or a substantial portion of a sorbitolbiosynthetic enzyme selected from the group consisting of aldehydereductase, NADP-dependent D-sorbitol-6-phosphate dehydrogenase orsorbitol dehydrogenase.

[0007] In another embodiment, the instant invention relates to achimeric gene encoding an aldehyde reductase, NADP-dependentD-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase, or to achimeric gene that comprises a nucleic acid fragment that iscomplementary to a nucleic acid fragment encoding an aldehyde reductase,NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitoldehydrogenase, operably linked to suitable regulatory sequences, whereinexpression of the chimeric gene results in production of levels of theencoded protein in a transformed host cell that is altered (i.e.,increased or decreased) from the level produced in an untransformed hostcell.

[0008] In a further embodiment, the instant invention concerns atransformed host cell comprising in its genome a chimeric gene encodingan aldehyde reductase, NADP-dependent D-sorbitol-6-phosphatedehydrogenase or sorbitol dehydrogenase, operably linked to suitableregulatory sequences. Expression of the chimeric gene results inproduction of altered levels of the encoded protein in the transformedhost cell. The transformed host cell can be of eukaryotic or prokaryoticorigin, and include cells derived from higher plants and microorganisms.The invention also includes transformed plants that arise fromtransformed host cells of higher plants, and seeds derived from suchtransformed plants.

[0009] An additional embodiment of the instant invention concerns amethod of altering the level of expression of an aldehyde reductase,NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitoldehydrogenase in a transformed host cell comprising: a) transforming ahost cell with a chimeric gene comprising a nucleic acid fragmentencoding an aldehyde reductase, NADP-dependent D-sorbitol-6-phosphatedehydrogenase or sorbitol dehydrogenase; and b) growing the transformedhost cell under conditions that are suitable for expression of thechimeric gene wherein expression of the chimeric gene results inproduction of altered levels of aldehyde reductase, NADP-dependentD-sorbitol-6-phosphate dehydrogenase or sorbitol dehydrogenase in thetransformed host cell.

[0010] An addition embodiment of the instant invention concerns a methodfor obtaining a nucleic acid fragment encoding all or a substantialportion of an amino acid sequence encoding an aldehyde reductase,NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitoldehydrogenase.

BRIEF DESCRIPTION OF THE SEQUENCE DESCRIPTIONS

[0011] The invention can be more fully understood from the followingdetailed description and the accompanying Sequence Listing which form apart of this application.

[0012] Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825. TABLE 1 Sorbitol Biosynthetic EnzymesSEQ ID NO: (Amino Protein Clone Designation (Nucleotide) Acid) Aldehydereductase Contig composed 1 2 of: ccase-b.pk0023.g2 ceb5.pk0015.g5ceb5.pk0032.e9 ceb5.pk0044.c8 ceb5.pk0049.d10 ceb5.pk0052.a4ceb5.pk0052.h8 ceb5.pk0062.a4 ceb5.pk0075.e7 ceb5.pk0075.g12cen6.pk0001.g2 Aldehyde reductase rca1n.pk022.j17 3 4 Aldehyde reductasesr1.pk0003.c5 5 6 Aldehyde reductase wl1n.pk0078.e5 7 8 NADP-dependentp0002.cgevj66r 9 10 D-sorbitol- 6-phosphate dehydrogenase NADP-dependentrls2.pk0004.b8 11 12 D-sorbitol- 6-phosphate dehydrogenaseNADP-dependent ses2w.pk0038.e12 13 14 D-sorbitol- 6-phosphatedehydrogenase Sorbitol dehydrogenase p0113.cieae77r 15 16 Sorbitoldehydrogenase rlr6.pk0096.b8 17 18 Sorbitol dehydrogenasesgs6c.pk001.122 19 20 Sorbitol dehydrogenase wlm96.pk0016.h12 21 22

[0013] The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Research 13:3021-3030 (1985) and in the BiochemicalJournal 219 (No. 2):345-373 (1984) which are herein incorporated byreference. The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

[0014] In the context of this disclosure, a number of terms shall beutilized. As used herein, a “nucleic acid fragment” is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. A nucleic acidfragment in the form of a polymer of DNA may be comprised of one or moresegments of cDNA, genomic DNA or synthetic DNA.

[0015] As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example, thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

[0016] As used herein, “substantially similar” refers to nucleic acidfragments wherein changes in one or more nucleotide bases results insubstitution of one or more amino acids, but do not affect thefunctional properties of the polypeptide encoded by the nucleotidesequence. “Substantially similar” also refers to nucleic acid fragmentswherein changes in one or more nucleotide bases does not affect theability of the nucleic acid fragment to mediate alteration of geneexpression by gene silencing through for example antisense orco-suppression technology. “Substantially similar” also refers tomodifications of the nucleic acid fragments of the instant inventionsuch as deletion or insertion of one or more nucleotides that do notsubstantially affect the functional properties of the resultingtranscript vis-à-vis the ability to mediate gene silencing or alterationof the functional properties of the resulting protein molecule. It istherefore understood that the invention encompasses more than thespecific exemplary nucleotide or amino acid sequences and includesfunctional equivalents thereof.

[0017] For example, it is well known in the art that antisensesuppression and co-suppression of gene expression may be accomplishedusing nucleic acid fragments representing less than the entire codingregion of a gene, and by nucleic acid fragments that do not share 100%sequence identity with the gene to be suppressed. Moreover, alterationsin a nucleic acid fragment which result in the production of achemically equivalent amino acid at a given site, but do not effect thefunctional properties of the encoded polypeptide, are well known in theart. Thus, a codon for the amino acid alanine, a hydrophobic amino acid,may be substituted by a codon encoding another less hydrophobic residue,such as glycine, or a more hydrophobic residue, such as valine, leucine,or isoleucine. Similarly, changes which result in substitution of onenegatively charged residue for another, such as aspartic acid forglutamic acid, or one positively charged residue for another, such aslysine for arginine, can also be expected to produce a functionallyequivalent product. Nucleotide changes which result in alteration of theN-terminal and C-terminal portions of the polypeptide molecule wouldalso not be expected to alter the activity of the polypeptide. Each ofthe proposed modifications is well within the routine skill in the art,as is determination of retention of biological activity of the encodedproducts.

[0018] Moreover, substantially similar nucleic acid fragments may alsobe characterized by their ability to hybridize. Estimates of suchhomology are provided by either DNA-DNA or DNA-RNA hybridization underconditions of stringency as is well understood by those skilled in theart (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRLPress, Oxford, U.K.). Stringency conditions can be adjusted to screenfor moderately similar fragments, such as homologous sequences fromdistantly related organisms, to highly similar fragments, such as genesthat duplicate functional enzymes from closely related organisms.Post-hybridization washes determine stringency conditions. One set ofpreferred conditions uses a series of washes starting with 6×SSC, 0.5%SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDSat 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at50° C. for 30 min. A more preferred set of stringent conditions useshigher temperatures in which the washes are identical to those aboveexcept for the temperature of the final two 30 min washes in 0.2×SSC,0.5% SDS was increased to 60° C. Another preferred set of highlystringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65°C.

[0019] Substantially similar nucleic acid fragments of the instantinvention may also be characterized by the percent identity of the aminoacid sequences that they encode to the amino acid sequences disclosedherein, as determined by algorithms commonly employed by those skilledin this art. Preferred are those nucleic acid fragments whose nucleotidesequences encode amino acid sequences that are 80% identical to theamino acid sequences reported herein. More preferred nucleic acidfragments encode amino acid sequences that are 90% identical to theamino acid sequences reported herein. Most preferred are nucleic acidfragments that encode amino acid sequences that are 95% identical to theamino acid sequences reported herein. Sequence alignments and percentidentity calculations were performed using the Megalign program of theLASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).Multiple alignment of the sequences was performed using the Clustalmethod of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) withthe default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the Clustal method were KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0020] A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more contiguous nucleotides isnecessary in order to putatively identify a polypeptide or nucleic acidsequence as homologous to a known protein or gene. Moreover, withrespect to nucleotide sequences, gene-specific oligonucleotide probescomprising 30 or more contiguous nucleotides may be used insequence-dependent methods of gene identification (e.g., Southernhybridization) and isolation (e.g., in situ hybridization of bacterialcolonies or bacteriophage plaques). In addition, short oligonucleotidesof 12 or more nucleotides may be used as amplification primers in PCR inorder to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises a nucleotide sequence that will afford specific identificationand/or isolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches amino acid and nucleotide sequencesencoding polypeptides that comprise one or more particular plantproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

[0021] “Codon degeneracy” refers to divergence in the genetic codepermitting variation of the nucleotide sequence without effecting theamino acid sequence of an encoded polypeptide. Accordingly, the instantinvention relates to any nucleic acid fragment comprising a nucleotidesequence that encodes all or a substantial portion of the amino acidsequences set forth herein. The skilled artisan is well aware of the“codon-bias” exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. Therefore, when synthesizing anucleic acid fragment for improved expression in a host cell, it isdesirable to design the nucleic acid fragment such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

[0022] “Synthetic nucleic acid fragments” can be assembled fromoligonucleotide building blocks that are chemically synthesized usingprocedures known to those skilled in the art. These building blocks areligated and annealed to form larger nucleic acid fragments which maythen be enzymatically assembled to construct the entire desired nucleicacid fragment. “Chemically synthesized”, as related to nucleic acidfragment, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of nucleic acid fragments may be accomplishedusing well established procedures, or automated chemical synthesis canbe performed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of nucleotide sequence to reflect thecodon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

[0023] “Gene” refers to a nucleic acid fragment that expresses aspecific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

[0024] “Coding sequence” refers to a nucleotide sequence that codes fora specific amino acid sequence. “Regulatory sequences” refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

[0025] “Promoter” refers to a nucleotide sequence capable of controllingthe expression of a coding sequence or functional RNA. In general, acoding sequence is located 3′ to a promoter sequence. The promotersequence consists of proximal and more distal upstream elements, thelatter elements often referred to as enhancers. Accordingly, an“enhancer” is a nucleotide sequence which can stimulate promoteractivity and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue-specificity of apromoter. Promoters may be derived in their entirety from a native gene,or be composed of different elements derived from different promotersfound in nature, or even comprise synthetic nucleotide segments. It isunderstood by those skilled in the art that different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions. Promoters which cause a nucleic acid fragmentto be expressed in most cell types at most times are commonly referredto as “constitutive promoters”. New promoters of various types useful inplant cells are constantly being discovered; numerous examples may befound in the compilation by Okamuro and Goldberg (1989) Biochemistry ofPlants 15:1-82. It is further recognized that since in most cases theexact boundaries of regulatory sequences have not been completelydefined, nucleic acid fragments of different lengths may have identicalpromoter activity.

[0026] The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) MolecularBiotechnology 3:225).

[0027] The “3′ non-coding sequences” refer to nucleotide sequenceslocated downstream of a coding sequence and include polyadenylationrecognition sequences and other sequences encoding regulatory signalscapable of affecting mRNA processing or gene expression. Thepolyadenylation signal is usually characterized by affecting theaddition of polyadenylic acid tracts to the 3′ end of the mRNAprecursor. The use of different 3′ non-coding sequences is exemplifiedby Ingelbrecht et al. (1989) Plant Cell 1:671-680.

[0028] “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptide by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to an RNAtranscript that includes the mRNA and so can be translated into apolypeptide by the cell. “Antisense RNA” refers to an RNA transcriptthat is complementary to all or part of a target primary transcript ormRNA and that blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

[0029] The term “operably linked” refers to the association of two ormore nucleic acid fragments on a single nucleic acid fragment so thatthe function of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

[0030] The term “expression”, as used herein, refers to thetranscription and stable accumulation of sense (mRNA) or antisense RNAderived from the nucleic acid fragment of the invention. Expression mayalso refer to translation of mRNA into a polypeptide. “Antisenseinhibition” refers to the production of antisense RNA transcriptscapable of suppressing the expression of the target protein.“Overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in normal ornon-transformed organisms. “Co-suppression” refers to the production ofsense RNA transcripts capable of suppressing the expression of identicalor substantially similar foreign or endogenous genes (U.S. Pat. No.5,231,020, incorporated herein by reference).

[0031] “Altered levels” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ from that ofnormal or non-transformed organisms.

[0032] “Mature” protein refers to a post-translationally processedpolypeptide; i.e., one from which any pre- or propeptides present in theprimary translation product have been removed. “Precursor” proteinrefers to the primary product of translation of mRNA; i.e., with pre-and propeptides still present. Pre- and propeptides may be but are notlimited to intracellular localization signals.

[0033] A “chloroplast transit peptide” is an amino acid sequence whichis translated in conjunction with a protein and directs the protein tothe chloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100: 1627-1632).

[0034] “Transformation” refers to the transfer of a nucleic acidfragment into the genome of a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” organisms. Examples ofmethods of plant transformation include Agrobacterium-mediatedtransformation (De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference).

[0035] Standard recombinant DNA and molecular cloning techniques usedherein are well known in the art and are described more fully inSambrook et al. Molecular Cloning. A Laboratory Manual; Cold SpringHarbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter“Maniatis”).

[0036] Nucleic acid fragments encoding at least a portion of severalsorbitol biosynthetic enzymes have been isolated and identified bycomparison of random plant cDNA sequences to public databases containingnucleotide and protein sequences using the BLAST algorithms well knownto those skilled in the art. The nucleic acid fragments of the instantinvention may be used to isolate cDNAs and genes encoding homologousproteins from the same or other plant species. Isolation of homologousgenes using sequence-dependent protocols is well known in the art.Examples of sequence-dependent protocols include, but are not limitedto, methods of nucleic acid hybridization, and methods of DNA and RNAamplification as exemplified by various uses of nucleic acidamplification technologies (e.g., polymerase chain reaction, ligasechain reaction).

[0037] For example, genes encoding other aldehyde reductase,NADP-dependent D-sorbitol-6-phosphate dehydrogenase or sorbitoldehydrogenase, either as cDNAs or genomic DNAs, could be isolateddirectly by using all or a portion of the instant nucleic acid fragmentsas DNA hybridization probes to screen libraries from any desired plantemploying methodology well known to those skilled in the art. Specificoligonucleotide probes based upon the instant nucleic acid sequences canbe designed and synthesized by methods known in the art (Maniatis).Moreover, the entire sequences can be used directly to synthesize DNAprobes by methods known to the skilled artisan such as random primer DNAlabeling, nick translation, or end-labeling techniques, or RNA probesusing available in vitro transcription systems. In addition, specificprimers can be designed and used to amplify a part or all of the instantsequences. The resulting amplification products can be labeled directlyduring amplification reactions or labeled after amplification reactions,and used as probes to isolate full length cDNA or genomic fragmentsunder conditions of appropriate stringency.

[0038] In addition, two short segments of the instant nucleic acidfragments may be used in polymerase chain reaction protocols to amplifylonger nucleic acid fragments encoding homologous genes from DNA or RNA.The polymerase chain reaction may also be performed on a library ofcloned nucleic acid fragments wherein the sequence of one primer isderived from the instant nucleic acid fragments, and the sequence of theother primer takes advantage of the presence of the polyadenylic acidtracts to the 3′ end of the mRNA precursor encoding plant genes.Alternatively, the second primer sequence may be based upon sequencesderived from the cloning vector. For example, the skilled artisan canfollow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci.USA 85:8998) to generate cDNAs by using PCR to amplify copies of theregion between a single point in the transcript and the 3′ or 5′ end.Primers oriented in the 3′ and 5′ directions can be designed from theinstant sequences. Using commercially available 3′ RACE or 5′ RACEsystems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Oharaet al. (1989) Proc. Natl. Acad. Sci. USA 86:5673; Loh et al. (1989)Science 243:217). Products generated by the 3′ and 5′ RACE procedurescan be combined to generate full-length cDNAs (Frohman and Martin (1989)Techniques 1:165).

[0039] Availability of the instant nucleotide and deduced amino acidsequences facilitates immunological screening of cDNA expressionlibraries. Synthetic peptides representing portions of the instant aminoacid sequences may be synthesized. These peptides can be used toimmunize animals to produce polyclonal or monoclonal antibodies withspecificity for peptides or proteins comprising the amino acidsequences. These antibodies can be then be used to screen cDNAexpression libraries to isolate full-length cDNA clones of interest(Lerner (1984) Adv. Immunol. 36: 1; Maniatis).

[0040] The nucleic acid fragments of the instant invention may be usedto create transgenic plants in which the disclosed polypeptides arepresent at higher or lower levels than normal or in cell types ordevelopmental stages in which they are not normally found. This wouldhave the effect of altering the level of sobitol biosynthesis in thosecells.

[0041] Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.For reasons of convenience, the chimeric gene may comprise promotersequences and translation leader sequences derived from the same genes.3′ Non-coding sequences encoding transcription termination signals mayalso be provided. The instant chimeric gene may also comprise one ormore introns in order to facilitate gene expression.

[0042] Plasmid vectors comprising the instant chimeric gene can thenconstructed. The choice of plasmid vector is dependent upon the methodthat will be used to transform host plants. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al. (1985) EMBO J.4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), andthus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, Western analysis of protein expression, or phenotypicanalysis.

[0043] For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by altering the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlaintPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100: 1627-1632) added and/or with targetingsequences that are already present removed. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of utility may be discovered in the future.

[0044] It may also be desirable to reduce or eliminate expression ofgenes encoding the instant polypeptides in plants for some applications.In order to accomplish this, a chimeric gene designed for co-suppressionof the instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

[0045] Molecular genetic solutions to the generation of plants withaltered gene expression have a decided advantage over more traditionalplant breeding approaches. Changes in plant phenotypes can be producedby specifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression ofspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

[0046] The person skilled in the art will know that specialconsiderations are associated with the use of antisense or cosuppresiontechnologies in order to reduce expression of particular genes. Forexample, the proper level of expression of sense or antisense genes mayrequire the use of different chimeric genes utilizing differentregulatory elements known to the skilled artisan. Once transgenic plantsare obtained by one of the methods described above, it will be necessaryto screen individual transgenics for those that most effectively displaythe desired phenotype. Accordingly, the skilled artisan will developmethods for screening large numbers of transformants. The nature ofthese screens will generally be chosen on practical grounds, and is notan inherent part of the invention. For example, one can screen bylooking for changes in gene expression by using antibodies specific forthe protein encoded by the gene being suppressed, or one could establishassays that specifically measure enzyme activity. A preferred methodwill be one which allows large numbers of samples to be processedrapidly, since it will be expected that a large number of transformantswill be negative for the desired phenotype.

[0047] The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to the these proteins by methodswell known to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded sorbitol biosynthetic enzyme. An example of a vector forhigh level expression of the instant polypeptides in a bacterial host isprovided (Example 8).

[0048] All or a substantial portion of the nucleic acid fragments of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

[0049] The production and use of plant gene-derived probes for use ingenetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol.Biol. Reporter 4(1):37-41. Numerous publications describe geneticmapping of specific cDNA clones using the methodology outlined above orvariations thereof. For example, F2 intercross populations, backcrosspopulations, randomly mated populations, near isogenic lines, and othersets of individuals may be used for mapping. Such methodologies are wellknown to those skilled in the art.

[0050] Nucleic acid probes derived from the instant nucleic acidsequences may also be used for physical mapping (i.e., placement ofsequences on physical maps; see Hoheisel et al. In: Nonmammalian GenomicAnalysis: A Practical Guide, Academic press 1996, pp. 319-346, andreferences cited therein).

[0051] In another embodiment, nucleic acid probes derived from theinstant nucleic acid sequences may be used in direct fluorescence insitu hybridization (FISH) mapping (Trask (1991) Trends Genet.7:149-154). Although current methods of FISH mapping favor use of largeclones (several to several hundred KB; see Laan et al. (1995) GenomeResearch 5:13-20), improvements in sensitivity may allow performance ofFISH mapping using shorter probes.

[0052] A variety of nucleic acid amplification-based methods of geneticand physical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 114(2):95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997)Nature Genetics 7:22-28) and Happy Mapping (Dear and Cook (1989) NucleicAcid Res. 17:6795-6807). For these methods, the sequence of a nucleicacid fragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

[0053] Loss of function mutant phenotypes may be identified for theinstant cDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402; Koes et al. (1995)Proc. Natl. Acad. Sci USA 92:8149; Bensen et al. (1995) Plant Cell7:75). The latter approach may be accomplished in two ways. First, shortsegments of the instant nucleic acid fragments may be used in polymerasechain reaction protocols in conjunction with a mutation tag sequenceprimer on DNAs prepared from a population of plants in which Mutatortransposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the instant polypeptides.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding theinstant polypeptides can be identified and obtained. This mutant plantcan then be used to determine or confirm the natural function of theinstant polypeptides disclosed herein.

EXAMPLES

[0054] The present invention is further defined in the followingExamples, in which all parts and percentages are by weight and degreesare Celsius, unless otherwise stated. It should be understood that theseExamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theinvention to adapt it to various usages and conditions.

Example 1

[0055] Composition of cDNA Libraries; Isolation and Sequencing of cDNAClones

[0056] cDNA libraries representing mRNAs from various corn, rice,soybean and wheat tissues were prepared. The characteristics of thelibraries are described below. TABLE 2 cDNA Libraries from Corn, Rice,Soybean and Wheat Library Tissue Clone ccase-b Corn (Zea mays L.) typeII callus tissue, somatic embryo ccase-b.pk0023.g2 formed ceb5 Corn (Zeamays L.) amplified embryo 30 day ceb5.pk0015.g5 ceb5.pk0032.e9ceb5.pk0044.c8 ceb5.pk0049.d10 ceb5.pk0052.a4 ceb5.pk0052.h8ceb5.pk0062.a4 ceb5.pk0075.e7 ceb5.pk0075.g12 cen6.pk0001.g2 p0002 Corn(Zea mays L.) tassel: premeiotic (>early p0002.cgevj66r uninucleate)p0113 Corn (Zea mays L.) intercalary meristem of expandingp0113.cieae77r internodes 5-9*; Sampled @ V10stage** rca1n Rice (Oryzasativa L.) callus* rca1n.pk022.j17 rlr6 Rice (Oryza sativa L.) leaf (15days after germination) 6 hrs rlr6.pk0096.b8 after infection ofMagaporthe grisea strain 4360-R-62 (AVR2-YAMO); Resistant rls2 Rice(Oryza sativa L.) leaf (15 DAG) 2 hrs after infection of rls2.pk0004.b8Magaporthe grisea strain 4360-R-67 (avr2-yamo); Susceptible ses2wSoybean (Glycine max L.) embryogenic suspension 2 weeks ses2w.pk0038.e12after subculture sgs6c Soybean (Glycine max L.) seeds 8 days aftergermination. sgs6c.pk001.122 sr1 Soybean (Glycine max L.) root librarysr1.pk0003.c5 wl1n Wheat (Triticum aestivum L.) leaf 7 day old seedling,light wl1n.pk0078.e5 grown* wlm96 Wheat (Triticum aestivum L.) seedlings96 hr after wlm96.pk0016.h12 inoculation w/E. graminis

[0057] cDNA libraries may be prepared by any one of many methodsavailable. For example, the cDNAs may be introduced into plasmid vectorsby first preparing the cDNA libraries in Uni-ZAP™ XR vectors accordingto the manufacturer's protocol (Stratagene Cloning Systems, La Jolla,Calif.). The Uni-ZAP™ XR libraries are converted into plasmid librariesaccording to the protocol provided by Stratagene. Upon conversion, cDNAinserts will be contained in the plasmid vector pBluescript. Inaddition, the cDNAs may be introduced directly into precut Bluescript IISK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs),followed by transfection into DH10B cells according to themanufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts arein plasmid vectors, plasmid DNAs are prepared from randomly pickedbacterial colonies containing recombinant pBluescript plasmids, or theinsert cDNA sequences are amplified via polymerase chain reaction usingprimers specific for vector sequences flanking the inserted cDNAsequences. Amplified insert DNAs or plasmid DNAs are sequenced indye-primer sequencing reactions to generate partial cDNA sequences(expressed sequence tags or “ESTs”; see Adams et al., (1991) Science252:1651). The resulting ESTs are analyzed using a Perkin Elmer Model377 fluorescent sequencer.

Example 2 Identification of cDNA Clones

[0058] cDNA clones encoding sorbitol biosynthetic enzymes wereidentified by conducting BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequencescontained in the BLAST “nr” database (comprising all non-redundantGenBank CDS translations, sequences derived from the 3-dimensionalstructure Brookhaven Protein Data Bank, the last major release of theSWISS-PROT protein sequence database, EMBL, and DDBJ databases). ThecDNA sequences obtained in Example 1 were analyzed for similarity to allpublicly available DNA sequences contained in the “nr” database usingthe BLASTN algorithm provided by the National Center for BioteclnologyInformation (NCBI). The DNA sequences were translated in all readingframes and compared for similarity to all publicly available proteinsequences contained in the “nr” database using the BLASTX algorithm(Gish and States (1993) Nature Genetics 3:266-272) provided by the NCBI.For convenience, the P-value (probability) of observing a match of acDNA sequence to a sequence contained in the searched databases merelyby chance as calculated by BLAST are reported herein as “pLog” values,which represent the negative of the logarithm of the reported P-value.Accordingly, the greater the pLog value, the greater the likelihood thatthe cDNA sequence and the BLAST “hit” represent homologous proteins.

Example 3 Characterization of cDNA Clones Encoding Aldehyde Reductase

[0059] The BLASTX search using the EST sequences from clones listed inTable 3 revealed similarity of the polypeptides encoded by the cDNAs toaldehyde reductase from Hordeum vulgare (NCBI Identifier No. gi 113595),Avena fatua (NCBI Identifier No. gi 2130022) and Medicago sativa (NCBIIdentifier No. gi 3378650). Shown in Table 3 are the BLAST results forindividual ESTs (“EST”), the sequences of the entire cDNA insertscomprising the indicated cDNA clones (“FIS”), or contigs assembled fromtwo or more ESTs (“Contig”): TABLE 3 BLAST Results for SequencesEncoding Polypeptides Homologous to Hordeum vulgare, Avena fatua andMedicago sativa Aldehyde Reductase Clone Status BLAST pLog Score Contigcomposed of: Contig 171.00 (gi 113595) ccase-b.pk0023.g2 ceb5.pk0015.g5ceb5.pk0032.e9 ceb5.pk0044.c8 ceb5.pk0049.d10 ceb5.pk0052.a4ceb5.pk0052.h8 ceb5.pk0062.a4 ceb5.pk0075.e7 ceb5.pk0075.g12cen6.pk0001.g2 rca1n.pk022.j17 FIS 172.00 (gi 2130022) sr1.pk0003.c5 FIS157.00 (gi 3378650) wl1n.pk0078.e5 EST 112.00 (gi 3378650)

[0060] The data in Table 4 represents a calculation of the percentidentity of the amino acid sequences set forth in SEQ ID NOs:2, 4, 6 and8 and the Hordeum vulgare, Avenafatua and Medicago sativa sequences (SEQID NOs:23, 24 and 25). TABLE 4 Percent Identity of Amino Acid SequencesDeduced From the Nucleotide Sequences of DNA Clones EncodingPolypeptides Homologous to Hordeum vulgare, Avena fatua and Medicagosativa Aldehyde Reductase SEQ ID NO. Percent Identity to 2 87% (gi113595) 4 90% (gi 2130022) 6 84% (gi 3378650) 8 61% (gi 3378650)

[0061] Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASARGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores andprobabilities indicate that the nucleic acid fragments comprising theinstant cDNA clones encode a substantial portion of an aldehydereductase. These sequences represent the first corn, rice, soybean andwheat sequences encoding aldehyde reductase.

Example 4 Characterization of cDNA Clones Encoding NADPH-DependentMannose 6-Phosphate Dehydrogense

[0062] The BLASTX search using the EST sequences from clones listed inTable 5 revealed similarity of the polypeptides encoded by the cDNAs toNADPH-dependent mannose 6-phosphate dehydrogense from Apium graveolens(NCBI Identifier No. gi 1835701). Shown in Table 5 are the BLAST resultsfor individual ESTs (“EST”), the sequences of the entire cDNA insertscomprising the indicated cDNA clones (“FIS”), or contigs assembled fromtwo or more ESTs (“Contig”): TABLE 5 BLAST Results for SequencesEncoding Polypeptides Homologous to Apium graveolens NADPH-DependentMannose 6-Phosphate Dehydrogense Clone Status BLAST pLog Score to (gi1835701) p0002.cgevj66r EST 134.00 rls2.pk0004.b8 FIS 138.00ses2w.pk0038.e12 EST 138.00

[0063] The data in Table 6 represents a calculation of the percentidentity of the amino acid sequences set forth in SEQ ID NOs:10, 12 and14 and the Apium graveolens sequences (SEQ ID NO:26). TABLE 6 PercentIdentity of Amino Acid Sequences Deduced From the Nucleotide Sequencesof DNA Clones Encoding Polypeptides Homologous to Apium graveolensNADPH-Dependent Mannose 6-Phosphate Dehydrogense SEQ ID NO. PercentIdentity to (gi 1835701) 10 70% 12 71% 14 74%

[0064] Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASARGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores andprobabilities indicate that the nucleic acid fragments comprising theinstant cDNA clones encode a substantial portion of a NADPH-dependentmannose 6-phosphate dehydrogense. These sequences represent the firstcorn, rice and soybean sequences encoding NADPH-dependent mannose6-phosphate dehydrogense.

Example 5 Characterization of cDNA Clones Encoding SorbitolDehydrogenase

[0065] The BLASTX search using the EST sequences from clones listed inTable 7 revealed similarity of the polypeptides encoded by the cDNAs tosorbitol dehydrogenase from Malus domestica (NCBI Identifier No. gi4519539). Shown in Table 7 are the BLAST results for individual ESTs(“EST”), the sequences of the entire cDNA inserts comprising theindicated cDNA clones (“FIS”), or contigs assembled from two or moreESTs (“Contig”): TABLE 7 BLAST Results for Sequences EncodingPolypeptides Homologous to Malus domestica Sorbitol Dehydrogenase CloneStatus BLAST pLog Score to (gi 4519539) p0113.cieae77r EST 158.00rlr6.pk0096.b8 EST  35.70 sgs6c.pk001.122 FIS 133.00 wlm96.pk0016.h12FIS 129.00

[0066] The data in Table 8 represents a calculation of the percentidentity of the amino acid sequences set forth in SEQ ID NOs:16, 18, 20and 22 and the Malus domestica sequence (SEQ ID NO:27). TABLE 8 PercentIdentity of Amino Acid Sequences Deduced From the Nucleotide Sequencesof DNA Clones Encoding Polypeptides Homologous to Malus domesticaSorbitol Dehydrogenase SEQ ID NO. Percent Identity to (gi 4519539)p0113.cieae77r 71% rlr6.pk0096.b8 45% sgs6c.pk001.122 69%wlm96.pk0016.h12 72%

[0067] Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASARGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores andprobabilities indicate that the nucleic acid fragments comprising theinstant cDNA clones encode a substantial portion of a sorbitoldehydrogenase. These sequences represent the first corn, rice, soybeanand wheat sequences encoding sorbitol dehydrogenase.

Example 6 Expression of Chimeric Genes in Monocot Cells

[0068] A chimeric gene comprising a cDNA encoding the instantpolypeptides in sense orientation with respect to the maize 27 kD zeinpromoter that is located 5′ to the cDNA fragment, and the 10 kD zein 3′end that is located 3′ to the cDNA fragment, can be constructed. ThecDNA fragment of this gene may be generated by polymerase chain reaction(PCR) of the cDNA clone using appropriate oligonucleotide primers.Cloning sites (NcoI or SmaI) can be incorporated into theoligonucleotides to provide proper orientation of the DNA fragment wheninserted into the digested vector pML103 as described below.Amplification is then performed in a standard PCR. The amplified DNA isthen digested with restriction enzymes NcoI and SmaI and fractionated onan agarose gel. The appropriate band can be isolated from the gel andcombined with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. PlasmidpML103 has been deposited under the terms of the Budapest Treaty at ATCC(American Type Culture Collection, 10801 University Blvd., Manassas, Va.20110-2209), and bears accession number ATCC 97366. The DNA segment frompML103 contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kDzein gene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insertDNA can be ligated at 15° C. overnight, essentially as described(Maniatis). The ligated DNA may then be used to transform E. coli XL1-Blue (Epicurian Coli XL-1 Blue™; Stratagene). Bacterial transformantscan be screened by restriction enzyme digestion of plasmid DNA andlimited nucleotide sequence analysis using the dideoxy chain terminationmethod (Sequenase™ DNA Sequencing Kit; U.S. Biochemical). The resultingplasmid construct would comprise a chimeric gene encoding, in the 5′ to3′ direction, the maize 27 kD zein promoter, a cDNA fragment encodingthe instant polypeptides, and the 10 kD zein 3′ region.

[0069] The chimeric gene described above can then be introduced intocorn cells by the following procedure. Immature corn embryos can bedissected from developing caryopses derived from crosses of the inbredcorn lines H99 and LH132. The embryos are isolated 10 to 11 days afterpollination when they are 1.0 to 1.5 mm long. The embryos are thenplaced with the axis-side facing down and in contact withagarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking18:659-668). The embryos are kept in the dark at 27° C. Friableembryogenic callus consisting of undifferentiated masses of cells withsomatic proembryoids and embryoids borne on suspensor structuresproliferates from the scutellum of these immature embryos. Theembryogenic callus isolated from the primary explant can be cultured onN6 medium and sub-cultured on this medium every 2 to 3 weeks.

[0070] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0071] The particle bombardment method (Klein et al. (1987) Nature327:70-73) may be used to transfer genes to the callus culture cells.According to this method, gold particles (1 μm in diameter) are coatedwith DNA using the following technique. Ten μg of plasmid DNAs are addedto 50 μL of a suspension of gold particles (60 mg per mL). Calciumchloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL ofa 1.0 M solution) are added to the particles. The suspension is vortexedduring the addition of these solutions. After 10 minutes, the tubes arebriefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed.The particles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

[0072] For bombardment, the embryogenic tissue is placed on filter paperover agarose-solidified N6 medium. The tissue is arranged as a thin lawnand covered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

[0073] Seven days after bombardment the tissue can be transferred to N6medium that contains gluphosinate (2 mg per liter) and lacks casein orproline. The tissue continues to grow slowly on this medium. After anadditional 2 weeks the tissue can be transferred to fresh N6 mediumcontaining gluphosinate. After 6 weeks, areas of about 1 cm in diameterof actively growing callus can be identified on some of the platescontaining the glufosinate-supplemented medium. These calli may continueto grow when sub-cultured on the selective medium.

[0074] Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 7 Expression of Chimeric Genes in Dicot Cells

[0075] A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), Sma I, Kpn I and Xba I.The entire cassette is flanked by Hind III sites.

[0076] The cDNA fragment of this gene may be generated by polymerasechain reaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

[0077] Soybean embroys may then be transformed with the expressionvector comprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

[0078] Soybean embryogenic suspension cultures can maintained in 35 mLliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

[0079] Soybean embryogenic suspension cultures may then be transformedby the method of particle gun bombardment (Klein et al. (1987) Nature(London) 327:70, U.S. Pat. No. 4,945,050). A DuPont Biolistic™PDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

[0080] A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

[0081] To 50 μL of a 60 mg/mL 1 λm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

[0082] Approximately 300-400 mg of a two-week-old suspension culture isplaced in an empty 60×15 mm petri dish and the residual liquid removedfrom the tissue with a pipette. For each transformation experiment,approximately 5-10 plates of tissue are normally bombarded. Membranerupture pressure is set at 1100 psi and the chamber is evacuated to avacuum of 28 inches mercury. The tissue is placed approximately 3.5inches away from the retaining screen and bombarded three times.Following bombardment, the tissue can be divided in half and placed backinto liquid and cultured as described above.

[0083] Five to seven days post bombardment, the liquid media may beexchanged with fresh media, and eleven to twelve days post bombardmentwith fresh media containing 50 mg/mL hygromycin. This selective mediacan be refreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 8 Expression of Chimeric Genes in Microbial Cells

[0084] The cDNAs encoding the instant polypeptides can be inserted intothe T7 E. coli expression vector pBT430. This vector is a derivative ofpET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

[0085] Plasmid DNA containing a cDNA may be appropriately digested torelease a nucleic acid fragment encoding the protein. This fragment maythen be purified on a 1% NuSieve GTG™ low melting agarose gel (FMC).Buffer and agarose contain 10 μg/ml ethidium bromide for visualizationof the DNA fragment. The fragment can then be purified from the agarosegel by digestion with GELase™ (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

[0086] For high level expression, a plasmid clone with the cDNA insertin the correct orientation relative to the T7 promoter can betransformed into E. coli strain BL21(DE3) (Studier et al. (1986) J. Mol.Biol. 189:113-130). Cultures are grown in LB medium containingampicillin (100 mg/L) at 25° C. At an optical density at 600 nm ofapproximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can beadded to a final concentration of 0.4 mM and incubation can be continuedfor 3 h at 25°. Cells are then harvested by centrifugation andre-suspended in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTTand 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glassbeads can be added and the mixture sonicated 3 times for about 5 secondseach time with a microprobe sonicator. The mixture is centrifuged andthe protein concentration of the supernatant determined. One μg ofprotein from the soluble fraction of the culture can be separated bySDS-polyacrylamide gel electrophoresis. Gels can be observed for proteinbands migrating at the expected molecular weight.

1 27 1 1297 DNA Zea mays 1 tttttttcaa gaagacagga gaagggagtt gtagtgagttttaagaatgg cgagtgcaca 60 ggcagtgggg caaggagaac gaggccactt cgttctgaagagcggacaca ccattccggc 120 cgttggtcta ggcacttgga gggccggctc agataccgctcactctgttc ggaccgccat 180 cgccgaggct ggatataggc acgtggacac agctgcccaatacggagtag agaaagaggt 240 cggtagagga cttaaagctg cgatggaggg cgggatcaacaggaaagatt tgtttgtgac 300 gtcgaagcta tggtgcaccg agctggctcc tgatagggttcggccagcac tcgagaaaac 360 actcaaggac ttgcagctgg attacctgga tctctaccttatccactggc ccttcaggct 420 gaaagacggg gcgcacatgc ccccggaagc cggggaggtgctggagttcg atatggaagg 480 ggtgtggagg gagatggaag gcctcgtgaa agacgggctcgtcaaggata taggtgtctg 540 caattacacg gtcgccaagc tcaaccgcct gatgcggtcagcgaatgttc caccggcagt 600 gtgccagatg gaaatgcacc ctgggtggaa gaacgacaggatctttgagg catgcaagaa 660 gcatgggatc catgttactg cttactctcc gctgggtccgtcagagaaga acctagcgca 720 cgacccgctc gtcgaaaagg tagccaacaa actggacaagaccccggggc aggtgctcct 780 caggtgggcg ctccagaggg ggacaagcgt cattcctaaatcgaccaagg acggaaggat 840 caaagagaac atccaggtgt tcgggtggga gatccctgaggaggacttca gggccctgtg 900 cggcatcaaa gatgagaagc gcgtgctgac cggagaggagctgttcgtga acaagaccca 960 cgggccgtac aagagcgcga ccgaggtgtg ggaccacgaggactgagcgg actgccgtgc 1020 cgccggatcc ctccactcca cctgatgaaa ccagaataaaggataccgac gcacctgtca 1080 gtcacctccc tcccgtgcct tgcgagagcg gcagcctctcgcacagggaa gatgctctgt 1140 gtctgagagc atgcagcctc gcacgagaaa gatgcagaaggagtgtgtgt ggcgcgcaat 1200 acactcctgt actgtacgat agactgaata ataataaagaagaaaacgca gcagtttgcc 1260 gttgcgtttt cctctgtgct tgcactatcg gtcgttc 12972 319 PRT Zea mays 2 Met Ala Ser Ala Gln Ala Val Gly Gln Gly Glu Arg GlyHis Phe Val 1 5 10 15 Leu Lys Ser Gly His Thr Ile Pro Ala Val Gly LeuGly Thr Trp Arg 20 25 30 Ala Gly Ser Asp Thr Ala His Ser Val Arg Thr AlaIle Ala Glu Ala 35 40 45 Gly Tyr Arg His Val Asp Thr Ala Ala Gln Tyr GlyVal Glu Lys Glu 50 55 60 Val Gly Arg Gly Leu Lys Ala Ala Met Glu Gly GlyIle Asn Arg Lys 65 70 75 80 Asp Leu Phe Val Thr Ser Lys Leu Trp Cys ThrGlu Leu Ala Pro Asp 85 90 95 Arg Val Arg Pro Ala Leu Glu Lys Thr Leu LysAsp Leu Gln Leu Asp 100 105 110 Tyr Leu Asp Leu Tyr Leu Ile His Trp ProPhe Arg Leu Lys Asp Gly 115 120 125 Ala His Met Pro Pro Glu Ala Gly GluVal Leu Glu Phe Asp Met Glu 130 135 140 Gly Val Trp Arg Glu Met Glu GlyLeu Val Lys Asp Gly Leu Val Lys 145 150 155 160 Asp Ile Gly Val Cys AsnTyr Thr Val Ala Lys Leu Asn Arg Leu Met 165 170 175 Arg Ser Ala Asn ValPro Pro Ala Val Cys Gln Met Glu Met His Pro 180 185 190 Gly Trp Lys AsnAsp Arg Ile Phe Glu Ala Cys Lys Lys His Gly Ile 195 200 205 His Val ThrAla Tyr Ser Pro Leu Gly Pro Ser Glu Lys Asn Leu Ala 210 215 220 His AspPro Leu Val Glu Lys Val Ala Asn Lys Leu Asp Lys Thr Pro 225 230 235 240Gly Gln Val Leu Leu Arg Trp Ala Leu Gln Arg Gly Thr Ser Val Ile 245 250255 Pro Lys Ser Thr Lys Asp Gly Arg Ile Lys Glu Asn Ile Gln Val Phe 260265 270 Gly Trp Glu Ile Pro Glu Glu Asp Phe Arg Ala Leu Cys Gly Ile Lys275 280 285 Asp Glu Lys Arg Val Leu Thr Gly Glu Glu Leu Phe Val Asn LysThr 290 295 300 His Gly Pro Tyr Lys Ser Ala Thr Glu Val Trp Asp His GluAsp 305 310 315 3 1277 DNA Oryza sativa 3 ggcacgagga aaggtgaagatagctaaacg gtgtgacaag caaggtaata gaaaggcgcg 60 atcatggcga gtgccaaggcgatggcgcag gatgagcatc actttgttct gaagagtggt 120 catgccatcc ctgcagttgggttaggcact tggagggccg gctcagatac tgctcactcc 180 gttcagacag ccatcactgaggctggatac aggcatgtag atacggctgc tcaatatgga 240 atagaacagg aggtcggcaaagggcttaaa gctgcgatgg aagctggaat caacaggaaa 300 gatttgtttg tgacgtcaaaaatatggtgc acaaacttgg ctcctgagag agttcgacca 360 gcattaaaga acacgctgaaggatctccag ttggattata tcgaccttta ccttattcat 420 tggcccttcc gtctaaaagatggagcacac cagcctcctg aggctgggga agtcttggag 480 tttgacatgg aggcagtatggagggaaatg gagagacttg tgacagatgg actggttaag 540 gacattggtg tctgcaatttctcagttacc aagctcaaca gactgttgca atcagctaat 600 attccacctg cagtatgccagatggaaatg caccctggtt ggaagaacaa taagattttc 660 gaggcctgca aaaaacatggaattcatgtt actgcctact ccccactggg ttcttctgaa 720 aagaaccttg cgcatgatccagttgtcgag aagatagcca acaagctgaa caagactcca 780 ggtcaagtgc tcatcaagtgggctctccaa aggggaacaa gcgttattcc aaaatcaact 840 aaagatgaaa ggattaaggagaatatgcag gtgtttggat gggagatccc tgaagaggac 900 ttccaggtct tgtgcggcatcaaagatgag aagcgagtcc tgacaggaga ggagctcttc 960 gtgaacaaga cccatgggccatacaagagt gcatctgagg tctgggataa cgaggactaa 1020 gctgccatgt ttcacccaaagatatggcaa ataatggtta tgatgttggt catgcacgaa 1080 ggagttatgt gatgttctccaagcactgtg acgaaaccag ctaactcagc acaagatgta 1140 ctactatgtg taaaactagtattgttctat gtgtgctctt cttcaagctt tgtgtaacca 1200 gcttctcagc acaagatgtacagccagtgc tgtaagataa ataaaagtat catggctttg 1260 ccgttgcact tgctact 12774 318 PRT Oryza sativa 4 Met Ala Ser Ala Lys Ala Met Ala Gln Asp Glu HisHis Phe Val Leu 1 5 10 15 Lys Ser Gly His Ala Ile Pro Ala Val Gly LeuGly Thr Trp Arg Ala 20 25 30 Gly Ser Asp Thr Ala His Ser Val Gln Thr AlaIle Thr Glu Ala Gly 35 40 45 Tyr Arg His Val Asp Thr Ala Ala Gln Tyr GlyIle Glu Gln Glu Val 50 55 60 Gly Lys Gly Leu Lys Ala Ala Met Glu Ala GlyIle Asn Arg Lys Asp 65 70 75 80 Leu Phe Val Thr Ser Lys Ile Trp Cys ThrAsn Leu Ala Pro Glu Arg 85 90 95 Val Arg Pro Ala Leu Lys Asn Thr Leu LysAsp Leu Gln Leu Asp Tyr 100 105 110 Ile Asp Leu Tyr Leu Ile His Trp ProPhe Arg Leu Lys Asp Gly Ala 115 120 125 His Gln Pro Pro Glu Ala Gly GluVal Leu Glu Phe Asp Met Glu Ala 130 135 140 Val Trp Arg Glu Met Glu ArgLeu Val Thr Asp Gly Leu Val Lys Asp 145 150 155 160 Ile Gly Val Cys AsnPhe Ser Val Thr Lys Leu Asn Arg Leu Leu Gln 165 170 175 Ser Ala Asn IlePro Pro Ala Val Cys Gln Met Glu Met His Pro Gly 180 185 190 Trp Lys AsnAsn Lys Ile Phe Glu Ala Cys Lys Lys His Gly Ile His 195 200 205 Val ThrAla Tyr Ser Pro Leu Gly Ser Ser Glu Lys Asn Leu Ala His 210 215 220 AspPro Val Val Glu Lys Ile Ala Asn Lys Leu Asn Lys Thr Pro Gly 225 230 235240 Gln Val Leu Ile Lys Trp Ala Leu Gln Arg Gly Thr Ser Val Ile Pro 245250 255 Lys Ser Thr Lys Asp Glu Arg Ile Lys Glu Asn Met Gln Val Phe Gly260 265 270 Trp Glu Ile Pro Glu Glu Asp Phe Gln Val Leu Cys Gly Ile LysAsp 275 280 285 Glu Lys Arg Val Leu Thr Gly Glu Glu Leu Phe Val Asn LysThr His 290 295 300 Gly Pro Tyr Lys Ser Ala Ser Glu Val Trp Asp Asn GluAsp 305 310 315 5 1073 DNA Glycine max 5 gcacgagcat ttctatttctaactaattat tgtgcttatt attattgttg agaaagaaaa 60 gaatggcaaa gttaataaaattctttgagt tgaacacagg ggccaagatt ccttctgttg 120 ggttaggcac ttggcaagctgagcctggtg ttgtagccaa agctgtcacc acagccattc 180 tggttggata caggcatattgattgtgctc aagcgtataa caatcaagca gagattggtt 240 ctgctcttaa gaagctttttgatgatggtg tggtgaagcg tgaggactta tggatcacct 300 ccaaactctg gtgttcagatcatgcttcag aagatgtgcc caaagcattg gataaaacat 360 tgcaggattt gcaacttgattaccttgacc tctatctgat ccactggcca gtgcgcatga 420 aaagcggatc agttggattcaagaaggaat atctcgatca accggacatt cccagcacat 480 ggaaagcaat ggaggcactctatgactcag gcaaggcaag agccatagga gttagcaatt 540 tctcttcaaa aaagcttcaagatctcatga atatagcaag agtgcctcct gctgttaacc 600 aagtggaatt gcacccaggatggcagcagc caaagctgca tgcattctgt gaatctaaag 660 gagttcatct gtctggatattctccactgg gctcaccagg agttctcaaa agtgacattc 720 ttaagaatcc tgttgtgatagagattgcag agaaattggg gaagacaccg gcacaagttg 780 cccttaggtg gggactgcaaacaggtcata gtgtgctgcc taagagcact aatgagtcca 840 gaatcaaggg aaactttgatgtgtttgact ggtctattcc agaagaagtg atggataagt 900 tctctgaaat taagcaggatagactaatta agggcacttt ttttgttgac gagacctatg 960 gtgcctttaa gaccgttgaagagctttggg atggtgaact ctgagcaata tgcttcatag 1020 agatgttgac aaatgcactgctcctcaagc agattgattc cgccttttac ctg 1073 6 313 PRT Glycine max 6 MetAla Lys Leu Ile Lys Phe Phe Glu Leu Asn Thr Gly Ala Lys Ile 1 5 10 15Pro Ser Val Gly Leu Gly Thr Trp Gln Ala Glu Pro Gly Val Val Ala 20 25 30Lys Ala Val Thr Thr Ala Ile Leu Val Gly Tyr Arg His Ile Asp Cys 35 40 45Ala Gln Ala Tyr Asn Asn Gln Ala Glu Ile Gly Ser Ala Leu Lys Lys 50 55 60Leu Phe Asp Asp Gly Val Val Lys Arg Glu Asp Leu Trp Ile Thr Ser 65 70 7580 Lys Leu Trp Cys Ser Asp His Ala Ser Glu Asp Val Pro Lys Ala Leu 85 9095 Asp Lys Thr Leu Gln Asp Leu Gln Leu Asp Tyr Leu Asp Leu Tyr Leu 100105 110 Ile His Trp Pro Val Arg Met Lys Ser Gly Ser Val Gly Phe Lys Lys115 120 125 Glu Tyr Leu Asp Gln Pro Asp Ile Pro Ser Thr Trp Lys Ala MetGlu 130 135 140 Ala Leu Tyr Asp Ser Gly Lys Ala Arg Ala Ile Gly Val SerAsn Phe 145 150 155 160 Ser Ser Lys Lys Leu Gln Asp Leu Met Asn Ile AlaArg Val Pro Pro 165 170 175 Ala Val Asn Gln Val Glu Leu His Pro Gly TrpGln Gln Pro Lys Leu 180 185 190 His Ala Phe Cys Glu Ser Lys Gly Val HisLeu Ser Gly Tyr Ser Pro 195 200 205 Leu Gly Ser Pro Gly Val Leu Lys SerAsp Ile Leu Lys Asn Pro Val 210 215 220 Val Ile Glu Ile Ala Glu Lys LeuGly Lys Thr Pro Ala Gln Val Ala 225 230 235 240 Leu Arg Trp Gly Leu GlnThr Gly His Ser Val Leu Pro Lys Ser Thr 245 250 255 Asn Glu Ser Arg IleLys Gly Asn Phe Asp Val Phe Asp Trp Ser Ile 260 265 270 Pro Glu Glu ValMet Asp Lys Phe Ser Glu Ile Lys Gln Asp Arg Leu 275 280 285 Ile Lys GlyThr Phe Phe Val Asp Glu Thr Tyr Gly Ala Phe Lys Thr 290 295 300 Val GluGlu Leu Trp Asp Gly Glu Leu 305 310 7 654 DNA Triticum aestivum unsure(404) unsure (438) unsure (453) unsure (469) unsure (474) unsure (497)unsure (502) unsure (526) unsure (528) unsure (530) unsure (541) unsure(569) unsure (632)..(633) unsure (639) unsure (653) 7 attaaagatggctgaatcct ttgttctcag taccggctcg aggatcccat cggttgggct 60 tggcgtatggcaaatacaac ctgacgctgc caacgacgcc atctacgctg ctgtcaaggc 120 tgggtatcggcatattgact gtgcagcagc atacaacaat gaggaggagg tgggcctggc 180 tttgaagaaattatttgaag atggtgtggt taagcgtgat gatttgttta tcacctctaa 240 gctatgggctgctaatcatg cacctgaaga tgtggaagag ggaatcgaca ccacacttca 300 agatttgcagcttgactact tgggacttgt acctcatcca tggtccaatc cgcatcaaaa 360 aaaggaactaacacgatgac cctgaaaact tctccctaca gatnccctgc tacatgggca 420 gcgatggagaattatacnat ccggcaaaac tcntgcaatc cgcgtgatna ctcncttgta 480 agaactcatgattgctnccg tncacaatgc cccacatcaa caagtnantn caccggttgg 540 nacaagaaactcggaacttc aatcaaggng tcacttcgat ccgcttagca ctgatcctga 600 taaggcattctaaaccatgg gccgtcaaaa tnnaaaacnc atcccacgga cang 654 8 308 PRT Triticumaestivum 8 Phe Val Leu Ser Thr Gly Ser Arg Ile Pro Ser Val Gly Leu GlyVal 1 5 10 15 Trp Gln Ile Gln Pro Asp Ala Ala Asn Asp Ala Ile Tyr AlaAla Val 20 25 30 Lys Ala Gly Tyr Arg His Ile Asp Cys Ala Ala Ala Tyr AsnAsn Glu 35 40 45 Glu Glu Val Gly Leu Ala Leu Lys Lys Leu Phe Glu Asp GlyVal Val 50 55 60 Lys Arg Asp Asp Leu Phe Ile Thr Ser Lys Leu Trp Ala AlaAsn His 65 70 75 80 Ala Pro Glu Asp Val Glu Glu Gly Ile Asp Thr Thr LeuGln Asp Leu 85 90 95 Gln Leu Asp Tyr Leu Asp Leu Tyr Leu Ile His Gly ProIle Arg Ile 100 105 110 Lys Lys Gly Thr Ser Thr Met Thr Pro Glu Asn PheLeu Pro Thr Asp 115 120 125 Ile Pro Ala Thr Trp Ala Ala Met Glu Lys LeuTyr Asp Ser Gly Lys 130 135 140 Ala Arg Ala Ile Gly Val Ser Asn Phe SerCys Lys Lys Leu His Asp 145 150 155 160 Leu Leu Ala Val Ala Arg Val ProPro Ala Val Asn Gln Val Glu Cys 165 170 175 His Pro Val Trp Gln Gln AspLys Leu Arg Lys Leu Cys Gln Ser Asn 180 185 190 Gly Val His Leu Ser AlaPhe Ser Pro Leu Gly Ser Pro Gly Ser Pro 195 200 205 Trp Ile Asn Gly ProSer Val Leu Lys Asn Pro Ile Val Val Ser Val 210 215 220 Ala Asp Lys LeuGln Lys Thr Pro Ala Gln Val Ala Leu Arg Trp Gly 225 230 235 240 Ile GlnMet Gly His Ser Val Leu Pro Lys Ser Ala Asn Glu Ser Arg 245 250 255 IleLys Glu Asn Ile Asp Ile Phe Gly Trp Ser Ile Pro Glu Asp Leu 260 265 270Met Ala Lys Phe Ser Glu Ile Lys Gln Val Arg Leu Leu Thr Ala Glu 275 280285 Phe Val Val His Pro Gln Ala Gly Tyr Asn Thr Leu Glu Asp Phe Trp 290295 300 Asp Gly Glu Ile 305 9 406 DNA Zea mays unsure (76) unsure (320)unsure (406) 9 tgccaacgag gcgaacagcc ggccaatcta gcatcagcgc gcggggtctgagagcagagc 60 ggcagggcgc catggnggca tcggtggcgc tgagcagcgg gcaccggatgccggcggtgg 120 ggctgggcgt gtggcggatg gagaaggcgg acatccgcgg cctcatccacacagcgctcc 180 gcgtcggcta ccgccacctg gactgcgccg ctgactacca gaacgaagctgaagttggtg 240 acgcgctcgc agaggcattc cagaccggac tcgtcaagcg ggaggacctgttcatcacaa 300 ccaagctgtg gaactcagan catgggcatg tgcttgaagc ctgcaaggacagcctgaaga 360 agctgcagct agactatctc gacctctacc tcatccattt cccagn 406 10308 PRT Zea mays 10 Ser Val Ala Leu Ser Ser Gly His Arg Met Pro Ala ValGly Leu Gly 1 5 10 15 Val Trp Arg Met Glu Lys Ala Asp Ile Arg Gly LeuIle His Thr Ala 20 25 30 Leu Arg Val Gly Tyr Arg His Leu Asp Cys Ala AlaAsp Tyr Gln Asn 35 40 45 Glu Ala Glu Val Gly Asp Ala Leu Ala Glu Ala PheGln Thr Gly Leu 50 55 60 Val Lys Arg Glu Asp Leu Phe Ile Thr Thr Lys LeuTrp Asn Ser Asp 65 70 75 80 His Gly His Val Leu Glu Ala Cys Lys Asp SerLeu Lys Lys Leu Gln 85 90 95 Leu Asp Tyr Leu Asp Leu Tyr Leu Ile His PhePro Val Ala Thr Arg 100 105 110 His Thr Gly Val Gly Thr Thr Ser Ser AlaLeu Gly Asp Asp Gly Val 115 120 125 Leu Asp Ile Asp Thr Thr Ile Ser LeuGlu Thr Thr Trp His Ala Met 130 135 140 Glu Glu Leu Val Ser Met Gly LeuVal Arg Ser Ile Gly Ile Ser Asn 145 150 155 160 Tyr Asp Ile Phe Leu ThrArg Asp Cys Leu Ala Tyr Ala Lys Ile Lys 165 170 175 Pro Ala Val Asn GlnIle Glu Thr His Pro Tyr Phe Gln Arg Asp Ser 180 185 190 Leu Val Lys PheCys Gln Lys His Gly Ile Cys Val Thr Ala His Thr 195 200 205 Pro Leu GlyGly Ser Thr Ala Asn Ala Glu Trp Phe Gly Thr Val Ser 210 215 220 Cys LeuAsp Asp Pro Val Ile Lys Ser Leu Ala Asp Lys Tyr Gly Lys 225 230 235 240Thr Pro Ala Gln Leu Val Leu Arg Trp Gly Leu Gln Arg Asp Thr Val 245 250255 Val Ile Pro Lys Thr Ser Lys Val Glu Arg Leu Gln Glu Asn Phe Asp 260265 270 Val Phe Gly Phe Asp Ile Ser Gly Glu Asp Met Glu Arg Met Lys Ala275 280 285 Ile Asp Arg Lys Tyr Arg Thr Asn Gln Pro Ala Lys Phe Trp GlyIle 290 295 300 Asp Leu Tyr Ala 305 11 1683 DNA Oryza sativa 11gcacgagctt cttcttctcg tctccgattc caacgaggcg gcggagcaga gcagaggcgc 60gggatggcgg cggcgcaggg gagcggagtg ccggcggcgc tggcgctgag cagcggccac 120acgatgccgt cggtggggtt gggcgtgtgg cggatggact cccccgccat ccgcgacctc 180atccactccg cactccgcat cggctaccgc cacttcgact gcgccgctga ttaccaaaac 240gaggctgaag ttggggatgc acttgcagag gcattccaaa ctggacttgt caagagggag 300gatcttttca tcacaaccaa gttgtggaac tcagatcatg ggcatgtggt tgaagcatgt 360aaggatagct tgaaaaagtt gcggctagat tatctagatc tctaccttat ccacttccca 420gtagctaccc gtcatactgg agttggtacg actgctagtg ctcttggtga tgatggtgtg 480ctagacattg ataccactat ctcattggag acaacatggc acgctatgga ggatcttgtt 540tccatgggac tggttcgcag cattgggatt agcaactatg acatattcct taccagagat 600tgtttggctt atgctaagat aaagcccgca gtgaatcaaa tcgagacaca tccctacttc 660cagcgcgact gtcttgtcaa gttctgccag aagcatggga tcttagtcac tgcccatacc 720cctctgggtg gctccactgc caatactgag tggtttgggt ccgtctcatg cctcgacgac 780cctgtcatca agtctctggc tgagaaatat ggcaagacac cggcgcagct ggtgctccgg 840tgggggcttc agaggaacac agtggtgatc cccaagacat ccaaggagga gagattgcag 900gagaactttg cggtcttcga tttcgccatc tccgacgagg acatggagaa gatgagatcc 960atcgaccgga agtaccgcac caaccagcct gccaagttct ggggaatcga cctgtttgct 1020tgatgaatta tgagtcttta gcacgaacaa taatgggggc ttttctactg tccctgggag 1080cttcttgtgc aatcattttt ctctgaactg aaacttcttg tgctgaagga tgaagttgtt 1140gggatcgtgt gaacttgaat tgttattcaa agggaaaaaa aaagtgtgaa cttgaattgt 1200atctatattt cgtgtatttg cgcttctgca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1260aaaaaaaaaa aaaaaaaaca aacaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaac 1320cccccggggg ggggccggga accaattccc cccaaaaggg ggccgtttaa ccccccccaa 1380ggggcggtct tttaaaaact ccgggagggg aaaaacccgg ggtttaccaa attaaccccc 1440ttgggaaaaa accccctttt cccaaatggg gtaaaaacca aaagggcccc caccattccc 1500cttcccaaaa atttgccaac cctaaaggga aatgggaacc ccccttgtac gggcaaataa 1560acccccgcgg gtttggggtt tcccccacac gggcccgtaa aacttgaaag cccctaagcg 1620ccggcccctt tgcgtttttt ccccctcctt ttccccacaa gtttgcccgg gtttccccga 1680cag 1683 12 308 PRT Oryza sativa 12 Ala Leu Ala Leu Ser Ser Gly His ThrMet Pro Ser Val Gly Leu Gly 1 5 10 15 Val Trp Arg Met Asp Ser Pro AlaIle Arg Asp Leu Ile His Ser Ala 20 25 30 Leu Arg Ile Gly Tyr Arg His PheAsp Cys Ala Ala Asp Tyr Gln Asn 35 40 45 Glu Ala Glu Val Gly Asp Ala LeuAla Glu Ala Phe Gln Thr Gly Leu 50 55 60 Val Lys Arg Glu Asp Leu Phe IleThr Thr Lys Leu Trp Asn Ser Asp 65 70 75 80 His Gly His Val Val Glu AlaCys Lys Asp Ser Leu Lys Lys Leu Arg 85 90 95 Leu Asp Tyr Leu Asp Leu TyrLeu Ile His Phe Pro Val Ala Thr Arg 100 105 110 His Thr Gly Val Gly ThrThr Ala Ser Ala Leu Gly Asp Asp Gly Val 115 120 125 Leu Asp Ile Asp ThrThr Ile Ser Leu Glu Thr Thr Trp His Ala Met 130 135 140 Glu Asp Leu ValSer Met Gly Leu Val Arg Ser Ile Gly Ile Ser Asn 145 150 155 160 Tyr AspIle Phe Leu Thr Arg Asp Cys Leu Ala Tyr Ala Lys Ile Lys 165 170 175 ProAla Val Asn Gln Ile Glu Thr His Pro Tyr Phe Gln Arg Asp Cys 180 185 190Leu Val Lys Phe Cys Gln Lys His Gly Ile Leu Val Thr Ala His Thr 195 200205 Pro Leu Gly Gly Ser Thr Ala Asn Thr Glu Trp Phe Gly Ser Val Ser 210215 220 Cys Leu Asp Asp Pro Val Ile Lys Ser Leu Ala Glu Lys Tyr Gly Lys225 230 235 240 Thr Pro Ala Gln Leu Val Leu Arg Trp Gly Leu Gln Arg AsnThr Val 245 250 255 Val Ile Pro Lys Thr Ser Lys Glu Glu Arg Leu Gln GluAsn Phe Ala 260 265 270 Val Phe Asp Phe Ala Ile Ser Asp Glu Asp Met GluLys Met Arg Ser 275 280 285 Ile Asp Arg Lys Tyr Arg Thr Asn Gln Pro AlaLys Phe Trp Gly Ile 290 295 300 Asp Leu Phe Ala 305 13 560 DNA Glycinemax unsure (106) unsure (461) unsure (495) unsure (551) 13 ggaagagagaaaaaccatgg caataacact caacaatggc ttcaagatgc ctatcattgg 60 attgggcgtgtggcgcatgg aaggaaacga aatcagggac ctaatntctc aattccatca 120 aaattggttatcgccatttt gattgtgctg ctgactacaa aaacgaagca gaagttggag 180 atgcgcttaaggaggctttt gatagtggcc ttgtgaagag agaggatctc ttcattacca 240 ccaagctttggaattctgat caaggccacg ttcttgaggc gtgtaaagac agtctcaaga 300 agcttcagttaacgtatcta gatttatatc ttgttcactt tcctgttgcc gtaaggatac 360 tggggttggtaatacttcta gtcctttggg tgatgatggg gcctggacat agtacaccat 420 tccctggaaacgacctggca tgcaatggaa gatcttgttt ntcggcttgt tcgcagcata 480 ggtcgcactatgtanttctg acagagatgt tacatattca agtaagctgc tgtaatcgat 540 gaactcatcancttcaggtg 560 14 309 PRT Glycine max 14 Met Ala Ile Thr Leu Asn Asn GlyPhe Lys Met Pro Ile Ile Gly Leu 1 5 10 15 Gly Val Trp Arg Met Glu GlyAsn Glu Ile Arg Asp Leu Ile Leu Asn 20 25 30 Ser Ile Lys Ile Gly Tyr ArgHis Phe Asp Cys Ala Ala Asp Tyr Lys 35 40 45 Asn Glu Ala Glu Val Gly AspAla Leu Lys Glu Ala Phe Asp Ser Gly 50 55 60 Leu Val Lys Arg Glu Asp LeuPhe Ile Thr Thr Lys Leu Trp Asn Ser 65 70 75 80 Asp Gln Gly His Val LeuGlu Ala Cys Lys Asp Ser Leu Lys Lys Leu 85 90 95 Gln Leu Thr Tyr Leu AspLeu Tyr Leu Val His Phe Pro Val Ala Val 100 105 110 Arg His Thr Gly ValGly Asn Thr Ser Ser Pro Leu Gly Asp Asp Gly 115 120 125 Val Leu Asp IleAsp Thr Thr Ile Ser Leu Glu Thr Thr Trp His Ala 130 135 140 Met Glu AspLeu Val Ser Ser Gly Leu Val Arg Ser Ile Gly Ile Ser 145 150 155 160 AsnTyr Asp Ile Phe Leu Thr Arg Asp Cys Leu Ala Tyr Ser Lys Ile 165 170 175Lys Pro Ala Val Asn Gln Ile Glu Thr His Pro Tyr Phe Gln Arg Asp 180 185190 Ser Leu Val Lys Phe Cys Gln Lys His Gly Ile Cys Val Thr Ala His 195200 205 Thr Pro Leu Gly Gly Ala Ala Ala Asn Ala Glu Trp Phe Gly Thr Val210 215 220 Ser Cys Leu Asp Asp Gln Val Leu Lys Gly Leu Ala Glu Lys TyrLys 225 230 235 240 Lys Thr Ala Ala Gln Ile Ser Leu Arg Trp Gly Ile GlnArg Asn Thr 245 250 255 Val Val Ile Pro Lys Ser Ser Lys Leu Glu Arg LeuLys Glu Asn Phe 260 265 270 Gln Val Phe Asp Phe Glu Leu Ser Lys Glu AspMet Glu Leu Ile Gly 275 280 285 Ser Ile Asp Arg Lys Tyr Arg Thr Asn GlnPro Ala Val Phe Trp Gly 290 295 300 Ile Asp Leu Tyr Ala 305 15 1154 DNAZea mays 15 ccacgcgtcc gctcatctgc gtacacggtc tccctcttcc tgtcagtagtagagtgagag 60 tgaggcagcg agtgggagac aaggggaaat ggggaaggga gcgcaagggagcgatgcggc 120 ggcggcgggc ggcgaggtgg aggagaacat ggcggcgtgg ctggttgccaagaacaccct 180 caagatcatg cccttcaagc tcccgcccgt cggcccttat gatgtccgcgtgcgcatgaa 240 agcagtgggg atttgcggca gcgatgtgca ctacctcagg gagatgcgcatcgcgcactt 300 cgtggtgaag gagccgatgg tgatcgggca cgagtgcgcg ggcgtggtcgaggaggtggg 360 cgccggcgtg acgcacctgt ccgtgggcga ccgcgtggcg ctggagccgggcgtcagctg 420 ctggcgctgc cgccactgca agggcgggcg gtacaaccct gtgcgaggaacatgaagttc 480 ttcgccaccc cgccggtgca cggctcgctg gcgaaccagg tggtgcacccggccgacctg 540 tgcttcaagc tccccgacgg ggtgagcctg gaggagggcg ccatgtgcgagccgctgagc 600 gtgggcgtgc acgcgtgccg ccgcgcgggg gtggggcccg agacgggcgtgctcgtggtg 660 ggcgccggcc ccatcggcct ggtgtcgctg ctagcggcgc gagccttcggcgcgccgcgc 720 gtggtggtcg tggacgtgga cgaccaccgc ctggccgtgg cccaggtcgctgggcgcgga 780 cgcggcggtg cgggtgtcgc cccgcgcgga ggacctggcg gacgaggtggagcgcatccg 840 cgcggccatg ggctcggaca tcgacgtcag cctggactgc gccgggttcagcaagaccat 900 gtcgacggcg ctggaggcga cgcggcccgg cgggaaggtg tgcctggtcgggatgggcca 960 caacgagatg acgctgcccc tgacggcggc ggcggcgcgg gaggtggacgtggtgggcgt 1020 gttccggtac aaggacacct ggccgctgtg catcgacttc ctgcgcagcggcaaggtgga 1080 cgtcaagccg ctcatcaccc accgcttcgg tttctcgcag cgggacgtggaggaggcctt 1140 cgaggtcagc gccc 1154 16 341 PRT Zea mays 16 Ala Ala AlaGly Gly Glu Val Glu Glu Asn Met Ala Ala Trp Leu Val 1 5 10 15 Ala LysAsn Thr Leu Lys Ile Met Pro Phe Lys Leu Pro Pro Val Gly 20 25 30 Pro TyrAsp Val Arg Val Arg Met Lys Ala Val Gly Ile Cys Gly Ser 35 40 45 Asp ValHis Tyr Leu Arg Glu Met Arg Ile Ala His Phe Val Val Lys 50 55 60 Glu ProMet Val Ile Gly His Glu Cys Ala Gly Val Val Glu Glu Val 65 70 75 80 GlyAla Gly Val Thr His Leu Ser Val Gly Asp Arg Val Ala Leu Glu 85 90 95 ProGly Val Ser Cys Trp Arg Cys Arg His Cys Lys Gly Gly Arg Tyr 100 105 110Asn Pro Val Arg Asn Met Lys Phe Phe Ala Thr Pro Pro Val His Gly 115 120125 Ser Leu Ala Asn Gln Val Val His Pro Ala Asp Leu Cys Phe Lys Leu 130135 140 Pro Asp Gly Val Ser Leu Glu Glu Gly Ala Met Cys Glu Pro Leu Ser145 150 155 160 Val Gly Val His Ala Cys Arg Arg Ala Gly Val Gly Pro GluThr Gly 165 170 175 Val Leu Val Val Gly Ala Gly Pro Ile Gly Leu Val SerLeu Leu Ala 180 185 190 Ala Arg Ala Phe Gly Ala Pro Arg Val Val Val ValAsp Val Asp Asp 195 200 205 His Arg Leu Ala Val Ala Arg Ser Leu Gly AlaAsp Ala Ala Val Arg 210 215 220 Val Ser Pro Arg Ala Glu Asp Leu Ala AspGlu Val Glu Arg Ile Arg 225 230 235 240 Ala Ala Met Gly Ser Asp Ile AspVal Ser Leu Asp Cys Ala Gly Phe 245 250 255 Ser Lys Thr Met Ser Thr AlaLeu Glu Ala Thr Arg Pro Gly Gly Lys 260 265 270 Val Cys Leu Val Gly MetGly His Asn Glu Met Thr Leu Pro Leu Ala 275 280 285 Ala Arg Glu Val AspVal Val Gly Val Phe Arg Tyr Lys Asp Thr Trp 290 295 300 Pro Leu Cys IleAsp Phe Leu Arg Ser Gly Lys Val Asp Val Lys Pro 305 310 315 320 Leu IleThr His Arg Phe Gly Phe Ser Gln Arg Asp Val Glu Glu Ala 325 330 335 PheGlu Val Ser Ala 340 17 718 DNA Oryza sativa unsure (313)..(314) unsure(351) unsure (418) unsure (470) unsure (514) unsure (519) unsure (524)unsure (527) unsure (537) unsure (548) unsure (618) unsure (631) unsure(635) unsure (646) unsure (680) unsure (691) unsure (693) unsure (702)unsure (706) unsure (717)..(718) 17 gtttaaacga gtgagtgagt gaagaggaggaagatgggga agggagggaa aggagccgag 60 gcggcggcgg cggcggtggc cggagccggtgaggaggaga acatggcggc gtggctggtg 120 gcgaagaaca ccctcaagat catgcccttcaagctcccgc cagttgggcc ttatgatgtc 180 cgtgtccgga tgaaggcagt gggcatctgcggcagcgacg tgcactacct cagggagatg 240 cgcattgcgc atttcgtggt gaaggagccgatggtgatcg ggcacgagtg cgccggcgtg 300 atagaggagg tcnngcagcg gcgtgaccacctcgccgtcg gcgaccgcgt nggcgctcga 360 gcccggcatc aactgctggc gctcaagcactgcaagggcg gccgctacaa cttctgcnaa 420 gacatgaatt cttcgccacc ctcccgttcacggttcctcg ccaacaaatn gtcacctgtg 480 atctgtgctc aagctccgga gaacttaacctgangaagng catntcnaac cctgacntgg 540 cttcaccntg cgcgcccact ccggcggaaacggggtctat tatggccggg ccatttgctg 600 tcacctctgc gccccctngg caacctctatntgcntgcaa accctnctgc attctggcca 660 ccctagttcg ccccagattn caggtgacgtngngatggga tntacntccg taaaagnn 718 18 159 PRT Oryza sativa UNSURE (94)UNSURE (129) UNSURE (146) 18 Met Gly Lys Gly Gly Lys Gly Ala Glu Ala AlaAla Ala Ala Val Ala 1 5 10 15 Gly Ala Gly Glu Glu Glu Asn Met Ala AlaTrp Leu Val Ala Lys Asn 20 25 30 Thr Leu Lys Ile Met Pro Phe Lys Leu ProPro Val Gly Pro Tyr Asp 35 40 45 Val Arg Val Arg Met Lys Ala Val Gly IleCys Gly Ser Asp Val His 50 55 60 Tyr Leu Arg Glu Met Arg Ile Ala His PheVal Val Lys Glu Pro Met 65 70 75 80 Val Ile Gly His Glu Cys Ala Gly ValIle Glu Glu Val Xaa Gln Arg 85 90 95 Arg Asp His Leu Ala Val Gly Asp ArgVal Gly Ala Arg Ala Arg His 100 105 110 Gln Leu Leu Ala Leu Lys His CysLys Gly Gly Arg Tyr Asn Phe Cys 115 120 125 Xaa Asp Met Asn Ser Ser ProPro Ser Arg Ser Arg Phe Leu Ala Asn 130 135 140 Lys Xaa Ser Pro Val IleCys Ala Gln Ala Pro Glu Asn Leu Thr 145 150 155 19 1610 DNA Glycine max19 ttcggcacga ggaaatgggt aaggggggaa tgtcaattga tgaacatgga gaaggcaaag 60aagagaatat ggctgcttgg cttgttggaa tgaacactct caagattcag cctttcaagc 120ttcctacttt gggaccccat gatgtcagag ttagaatgaa ggctgttggt atctgtggga 180gtgatgttca ctacctcaag acactgaggt gtgctcactt tatagttaaa gaaccaatgg 240ttattggtca tgagtgtgct gggatcatag aagaagttgg tagtcaggta aagagtttgg 300tgcctggtga ccgtgtggca attgagcctg ggatcagttg ttggcattgc aaccattgca 360aacacggtcg atataactta tgcgatgata tgaagttttt tgctactcca ccagttcatg 420gttccctggc taatcagata gtgcatcctg cagacctatg ttttaagctg ccagacaacg 480tgagcctaga ggagggagca atgtgtgaac ccttaagtgt tggtgttcat gcttgtagaa 540gagctaatat tggaccagaa acaaatgtgt tgatcatggg agcagggccc ataggacttg 600ttacaatgct ggcagctcgt gcgtttgggg cacccaaaac agtcattgtg gatgttgatg 660accatcgttt atctgttgca aaatctcttg gtgcagatga tattattaaa gtctcaacaa 720acattaagga tgtggctgaa gaagttgtgc agatacagaa ggttatggga gctggtatag 780atgttacctt tgattgtgct ggttttgaca aaaccatgtc tccagcactg agtgctactc 840agccaggtgg caaagtttgc ctagtgggaa tgggacattc tgaaatgact gtcccactca 900cccccagctg cagcaagttg tattggattt tcatcacatt tttaaaggct tggatacttc 960actgatacat accgctgata tatccaagga agttgatgtg gttggagttt ttcgctatat 1020gaacacatgg cctctttgcc ttgagtttct aaggagtggc aaaattgatg tgaaacccct 1080tataacgcac aggtttggat tctctcaaaa ggaagtggaa gaagcctttg aaacaactgc 1140tcgtggtggt aacgccatca aggtcatgtt caatctttag atactggact gtgactttac 1200aaactgtcgt atgttgaaca aagtaggtaa tttacatgtt catgctcatg ttaaatacca 1260atgtattgat aacacgggtt atgaataaag tggtttcaag aaggacttgt aaaacatgtt 1320aatggtaact gcagcatcta cagattcagt tatgaagtga aactttttct taatatgtta 1380atgagtaaac cctcaaattt gtctagtagt aattgagaat ttcaatgcac aataacaaac 1440tgttgcttcc aacccgcttc atcatcactc tccctaatcc tacttctatt ctcctccttc 1500ccctctgcct cgcgtctgtt cgctcctact cgcttccttg tctccaccta tgattacatg 1560ataccttccc ctcggggggg ggggccgggc cacatatccc ccccaaagtg 1610 20 316 PRTGlycine max 20 Met Gly Lys Gly Gly Met Ser Ile Asp Glu His Gly Glu GlyLys Glu 1 5 10 15 Glu Asn Met Ala Ala Trp Leu Val Gly Met Asn Thr LeuLys Ile Gln 20 25 30 Pro Phe Lys Leu Pro Thr Leu Gly Pro His Asp Val ArgVal Arg Met 35 40 45 Lys Ala Val Gly Ile Cys Gly Ser Asp Val His Tyr LeuLys Thr Leu 50 55 60 Arg Cys Ala His Phe Ile Val Lys Glu Pro Met Val IleGly His Glu 65 70 75 80 Cys Ala Gly Ile Ile Glu Glu Val Gly Ser Gln ValLys Ser Leu Val 85 90 95 Pro Gly Asp Arg Val Ala Ile Glu Pro Gly Ile SerCys Trp His Cys 100 105 110 Asn His Cys Lys His Gly Arg Tyr Asn Leu CysAsp Asp Met Lys Phe 115 120 125 Phe Ala Thr Pro Pro Val His Gly Ser LeuAla Asn Gln Ile Val His 130 135 140 Pro Ala Asp Leu Cys Phe Lys Leu ProAsp Asn Val Ser Leu Glu Glu 145 150 155 160 Gly Ala Met Cys Glu Pro LeuSer Val Gly Val His Ala Cys Arg Arg 165 170 175 Ala Asn Ile Gly Pro GluThr Asn Val Leu Ile Met Gly Ala Gly Pro 180 185 190 Ile Gly Leu Val ThrMet Leu Ala Ala Arg Ala Phe Gly Ala Pro Lys 195 200 205 Thr Val Ile ValAsp Val Asp Asp His Arg Leu Ser Val Ala Lys Ser 210 215 220 Leu Gly AlaAsp Asp Ile Ile Lys Val Ser Thr Asn Ile Lys Asp Val 225 230 235 240 AlaGlu Glu Val Val Gln Ile Gln Lys Val Met Gly Ala Gly Ile Asp 245 250 255Val Thr Phe Asp Cys Ala Gly Phe Asp Lys Thr Met Ser Pro Ala Leu 260 265270 Ser Ala Thr Gln Pro Gly Gly Lys Val Cys Leu Val Gly Met Gly His 275280 285 Ser Glu Met Thr Val Pro Leu Thr Pro Ser Cys Ser Lys Leu Tyr Trp290 295 300 Ile Phe Ile Thr Phe Leu Lys Ala Trp Ile Leu His 305 310 31521 933 DNA Triticum aestivum 21 gcacgaggcg gccgtggccg gcgagggggagaacatggcg gcgtggctcg tggccaagaa 60 caccctcaag atcatgccct tcaagcttccgccgctgggt ccttatgatg tgcgagtccg 120 gatgaaggcg gtgggcatct gcggcagcgacgtgcattac ctcaaggaga tgcgcattgc 180 gcatttcgtg gtgaaagagc cgatggtgatcgggcacgag tgcgctggca tcatcgagga 240 ggtgggcgac ggcgtgaagc acctcgccgtgggagaccgc gtggcgctgg agcccggcat 300 cagctgctgg cgctgcaggc actgcaagggcggccgctac aacctctgcg acgacatgaa 360 gttcttcgcc accccacctt accatggatcacttgccgac cagattgtgc atccaggtga 420 cctgtgcttc aagcttccag acaacgtgagcctggaggag ggcgccatgt gcgagcccct 480 gagcgtgggg gtgcacgcct gccgccgagccgacgtgggc gcggagaaga gcgtgctcat 540 catgggcgcc ggcccgatcg gcctggtcaccatgctctcg gcgcgcgcct tcggggcgcc 600 caggatcgtc atcgccgacg tcgacgaccaccgcctctcc gtggccaagt ccctcggcgc 660 ggacgccgtc gtgaaggtct ccggcaacacggaggacctc gcgggggaga tcgagcgcat 720 ccaggcggcg atgggaggcg acatcgacgtgagcctggac tgcgccgggt tcagcaagac 780 gatgtcgacg gcgctggagg cgacgcggccgggcgggagg gtgtgcctgg tggggatggg 840 gcacaacgag atgacggtgc cgctgacgtcggcggcgatc cgggaggtgg acgtggtggg 900 gatcttccgt tacaaggaca cgtggccgctgtg 933 22 301 PRT Triticum aestivum 22 Glu Asn Met Ala Ala Trp Leu ValAla Lys Asn Thr Leu Lys Ile Met 1 5 10 15 Pro Phe Lys Leu Pro Pro LeuGly Pro Tyr Asp Val Arg Val Arg Met 20 25 30 Lys Ala Val Gly Ile Cys GlySer Asp Val His Tyr Leu Lys Glu Met 35 40 45 Arg Ile Ala His Phe Val ValLys Glu Pro Met Val Ile Gly His Glu 50 55 60 Cys Ala Gly Ile Ile Glu GluVal Gly Asp Gly Val Lys His Leu Ala 65 70 75 80 Val Gly Asp Arg Val AlaLeu Glu Pro Gly Ile Ser Cys Trp Arg Cys 85 90 95 Arg His Cys Lys Gly GlyArg Tyr Asn Leu Cys Asp Asp Met Lys Phe 100 105 110 Phe Ala Thr Pro ProTyr His Gly Ser Leu Ala Asp Gln Ile Val His 115 120 125 Pro Gly Asp LeuCys Phe Lys Leu Pro Asp Asn Val Ser Leu Glu Glu 130 135 140 Gly Ala MetCys Glu Pro Leu Ser Val Gly Val His Ala Cys Arg Arg 145 150 155 160 AlaAsp Val Gly Ala Glu Lys Ser Val Leu Ile Met Gly Ala Gly Pro 165 170 175Ile Gly Leu Val Thr Met Leu Ser Ala Arg Ala Phe Gly Ala Pro Arg 180 185190 Ile Val Ile Ala Asp Val Asp Asp His Arg Leu Ser Val Ala Lys Ser 195200 205 Leu Gly Ala Asp Ala Val Val Lys Val Ser Gly Asn Thr Glu Asp Leu210 215 220 Ala Gly Glu Ile Glu Arg Ile Gln Ala Ala Met Gly Gly Asp IleAsp 225 230 235 240 Val Ser Leu Asp Cys Ala Gly Phe Ser Lys Thr Met SerThr Ala Leu 245 250 255 Glu Ala Thr Arg Pro Gly Gly Arg Val Cys Leu ValGly Met Gly His 260 265 270 Asn Glu Met Thr Val Pro Leu Thr Ser Ala AlaIle Arg Glu Val Asp 275 280 285 Val Val Gly Ile Phe Arg Tyr Lys Asp ThrTrp Pro Leu 290 295 300 23 320 PRT Hordeum vulgare 23 Met Ala Ser AlaLys Ala Thr Met Gly Gln Gly Glu Gln Asp His Phe 1 5 10 15 Val Leu LysSer Gly His Ala Met Pro Ala Val Gly Leu Gly Thr Trp 20 25 30 Arg Ala GlySer Asp Thr Ala His Ser Val Arg Thr Ala Ile Thr Glu 35 40 45 Ala Gly TyrArg His Val Asp Thr Ala Ala Glu Tyr Gly Val Glu Lys 50 55 60 Glu Val GlyLys Gly Leu Lys Ala Ala Met Glu Ala Gly Ile Asp Arg 65 70 75 80 Lys AspLeu Phe Val Thr Ser Lys Ile Trp Cys Thr Asn Leu Ala Pro 85 90 95 Glu ArgVal Arg Pro Ala Leu Glu Asn Thr Leu Lys Asp Leu Gln Leu 100 105 110 AspTyr Ile Asp Leu Tyr His Ile His Trp Pro Phe Arg Leu Lys Asp 115 120 125Gly Ala His Met Pro Pro Glu Ala Gly Glu Val Leu Glu Phe Asp Met 130 135140 Glu Gly Val Trp Lys Glu Met Glu Asn Leu Val Lys Asp Gly Leu Val 145150 155 160 Lys Asp Ile Gly Val Cys Asn Tyr Thr Val Thr Lys Leu Asn ArgLeu 165 170 175 Leu Arg Ser Ala Lys Ile Pro Pro Ala Val Cys Gln Met GluMet His 180 185 190 Pro Gly Trp Lys Asn Asp Lys Ile Phe Glu Ala Cys LysLys His Gly 195 200 205 Ile His Val Thr Ala Tyr Ser Pro Leu Gly Ser SerGlu Lys Asn Leu 210 215 220 Ala His Asp Pro Val Val Glu Lys Val Ala AsnLys Leu Asn Lys Thr 225 230 235 240 Pro Gly Gln Val Leu Ile Lys Trp AlaLeu Gln Arg Gly Thr Ser Val 245 250 255 Ile Pro Lys Ser Ser Lys Asp GluArg Ile Lys Glu Asn Ile Gln Val 260 265 270 Phe Gly Trp Glu Ile Pro GluGlu Asp Phe Lys Val Leu Cys Ser Ile 275 280 285 Lys Asp Glu Lys Arg ValLeu Thr Gly Glu Glu Leu Phe Val Asn Lys 290 295 300 Thr His Gly Pro TyrArg Ser Ala Ala Asp Val Trp Asp His Glu Asn 305 310 315 320 24 319 PRTAvena fatua 24 Met Ala Ser Ala Lys Ala Met Gly Gln Gly Glu Gln Asp ArgPhe Val 1 5 10 15 Leu Lys Ser Gly His Ala Ile Pro Ala Val Gly Leu GlyThr Trp Arg 20 25 30 Ala Gly Ser Asp Thr Ala His Ser Val Gln Thr Ala IleThr Glu Ala 35 40 45 Gly Tyr Arg His Val Asp Thr Ala Ala Gln Tyr Gly IleGlu Lys Glu 50 55 60 Val Asp Lys Gly Leu Lys Ala Ala Met Glu Ala Gly IleAsp Arg Lys 65 70 75 80 Asp Leu Phe Val Thr Ser Lys Ile Trp Arg Thr AsnLeu Ala Pro Glu 85 90 95 Arg Ala Arg Pro Ala Leu Glu Asn Thr Leu Lys AspLeu Gln Leu Asp 100 105 110 Tyr Ile Asp Leu Tyr Leu Ile His Trp Pro PheArg Leu Lys Asp Gly 115 120 125 Ala His Gln Pro Pro Glu Ala Gly Glu ValLeu Glu Phe Asp Met Glu 130 135 140 Gly Val Trp Lys Glu Met Glu Lys LeuVal Lys Asp Gly Leu Val Lys 145 150 155 160 Asp Ile Asp Val Cys Asn PheThr Val Thr Lys Leu Asn Arg Leu Leu 165 170 175 Arg Ser Ala Asn Ile ProPro Ala Val Cys Gln Met Glu Met His Pro 180 185 190 Gly Trp Lys Asn AspLys Ile Phe Glu Ala Cys Lys Lys His Gly Ile 195 200 205 His Val Thr AlaTyr Ser Pro Leu Gly Ser Ser Glu Lys Asn Leu Val 210 215 220 His Asp ProVal Val Glu Lys Val Ala Asn Lys Leu Asn Lys Thr Pro 225 230 235 240 GlyGln Val Leu Ile Lys Trp Ala Leu Gln Arg Gly Thr Ser Val Ile 245 250 255Pro Lys Ser Ser Lys Asp Glu Arg Ile Lys Glu Asn Ile Gln Ala Phe 260 265270 Gly Trp Glu Ile Pro Glu Asp Asp Phe Gln Val Leu Cys Ser Ile Lys 275280 285 Asp Glu Lys Arg Val Leu Thr Gly Glu Glu Leu Phe Val Asn Lys Thr290 295 300 His Gly Pro Tyr Lys Ser Ala Ser Glu Val Trp Asp His Glu Asn305 310 315 25 313 PRT Medicago sativa 25 Met Ala Thr Ala Ile Lys PhePhe Gln Leu Asn Thr Gly Ala Lys Ile 1 5 10 15 Pro Ser Val Gly Leu GlyThr Trp Gln Ala Glu Pro Gly Val Val Ala 20 25 30 Lys Ala Val Thr Thr AlaVal Gln Val Gly Tyr Arg His Ile Asp Cys 35 40 45 Ala Glu Ala Tyr Lys AsnGln Ser Glu Ile Gly Ser Ala Leu Lys Lys 50 55 60 Leu Cys Glu Asp Gly ValVal Lys Arg Glu Glu Leu Trp Ile Thr Ser 65 70 75 80 Lys Leu Trp Cys SerAsp His His Pro Glu Asp Val Pro Lys Ala Leu 85 90 95 Asp Lys Thr Leu AsnAsp Leu Gln Leu Asp Tyr Leu Asp Leu Tyr Leu 100 105 110 Ile His Trp ProVal Ser Met Lys Arg Gly Thr Gly Glu Phe Met Gly 115 120 125 Glu Asn LeuAsp His Ala Asp Ile Pro Ser Thr Trp Lys Ala Leu Gly 130 135 140 Ala LeuTyr Asp Ser Gly Lys Ala Lys Ala Ile Gly Val Ser Asn Phe 145 150 155 160Ser Thr Lys Lys Leu Gln Asp Leu Leu Asp Val Ala Arg Val Pro Pro 165 170175 Ala Val Asn Gln Val Glu Leu His Pro Gly Trp Gln Gln Ala Lys Leu 180185 190 His Ala Phe Cys Glu Ser Lys Gly Ile His Leu Ser Gly Tyr Ser Pro195 200 205 Leu Gly Ser Pro Gly Val Leu Lys Ser Asp Ile Leu Lys Asn ProVal 210 215 220 Val Lys Glu Ile Ala Glu Lys Leu Gly Lys Thr Pro Gly GlnVal Ala 225 230 235 240 Leu Arg Trp Gly Leu Gln Ala Gly His Ser Val LeuPro Lys Ser Thr 245 250 255 Asn Glu Ala Arg Ile Lys Lys Asn Leu Asp ValTyr Asp Trp Ser Ile 260 265 270 Pro Glu Asp Leu Phe Pro Lys Phe Ser GluIle Lys Gln Asp Lys Leu 275 280 285 Ile Lys Gly Thr Phe Phe Val Asn AspThr Tyr Gly Ala Phe Arg Thr 290 295 300 Ile Glu Glu Leu Trp Asp Gly GluVal 305 310 26 309 PRT Apium graveolens 26 Met Ala Ile Thr Leu Asn SerGly Phe Lys Met Pro Val Leu Gly Leu 1 5 10 15 Gly Val Trp Arg Met AspArg Asn Glu Ile Lys Asn Leu Leu Leu Ser 20 25 30 Ala Ile Asn Leu Gly TyrArg His Phe Asp Cys Ala Ala Asp Tyr Lys 35 40 45 Asn Glu Leu Glu Val GlyGlu Ala Phe Lys Glu Ala Phe Asp Thr Asp 50 55 60 Leu Val Lys Arg Glu AspLeu Phe Ile Thr Thr Lys Leu Trp Asn Ser 65 70 75 80 Asp His Gly His ValIle Glu Ala Cys Lys Asn Ser Leu Lys Lys Leu 85 90 95 Gln Leu Glu Tyr LeuAsp Leu Tyr Leu Ile His Phe Pro Met Ala Ser 100 105 110 Lys His Ser GlyIle Gly Thr Thr Arg Ser Ile Leu Asp Asp Glu Gly 115 120 125 Val Trp GluVal Asp Ala Thr Ile Ser Leu Glu Ala Thr Trp His Glu 130 135 140 Met GluLys Leu Val Glu Met Gly Leu Val Arg Ser Ile Gly Ile Ser 145 150 155 160Asn Tyr Asp Val Tyr Leu Thr Arg Asp Ile Leu Ser Tyr Ser Lys Ile 165 170175 Lys Pro Ala Val Asn Gln Ile Glu Thr His Pro Tyr Phe Gln Arg Asp 180185 190 Ser Leu Ile Lys Phe Cys Gln Lys Tyr Gly Ile Ala Ile Thr Ala His195 200 205 Thr Pro Leu Gly Gly Ala Leu Ala Asn Thr Glu Arg Phe Gly SerVal 210 215 220 Ser Cys Leu Asp Asp Pro Val Leu Lys Lys Leu Ser Asp LysHis Asn 225 230 235 240 Lys Ser Pro Ala Gln Ile Val Leu Arg Trp Gly ValGln Arg Asn Thr 245 250 255 Ile Val Ile Pro Lys Ser Ser Lys Thr Lys ArgLeu Glu Glu Asn Ile 260 265 270 Asn Ile Phe Asp Phe Glu Leu Ser Lys GluAsp Met Glu Leu Ile Lys 275 280 285 Thr Met Glu Arg Asn Gln Arg Ser AsnThr Pro Ala Lys Ala Trp Gly 290 295 300 Ile Asp Val Tyr Ala 305 27 371PRT Malus domestica 27 Met Gly Lys Gly Gly Met Ser Asp Gly Asp His AlaAsp Arg Cys Cys 1 5 10 15 Gly Glu Ala Ile Asn Gly Asp Val Gln Gln GluAsn Met Ala Ala Trp 20 25 30 Leu Leu Gly Val Lys Asn Leu Lys Ile Gln ProTyr Lys Leu Pro Asn 35 40 45 Leu Gly Pro His Asp Val Arg Val Arg Leu ArgAla Val Gly Ile Cys 50 55 60 Gly Ser Asp Val His His Phe Lys Asn Met ArgCys Val Asp Phe Ile 65 70 75 80 Val Lys Glu Pro Met Val Ile Gly His GluCys Ala Gly Ile Ile Glu 85 90 95 Glu Val Gly Ser Glu Val Glu His Leu ValPro Gly Asp Arg Val Ala 100 105 110 Leu Glu Pro Gly Ile Ser Cys Lys ArgCys Asn Leu Cys Lys Gln Gly 115 120 125 Arg Tyr Asn Leu Cys Arg Lys MetLys Phe Phe Gly Ser Pro Pro Asn 130 135 140 Asn Gly Cys Leu Ala Asn GlnVal Val His Pro Gly Asp Leu Cys Phe 145 150 155 160 Lys Leu Pro Asp AsnVal Ser Leu Glu Glu Gly Ala Met Cys Glu Pro 165 170 175 Leu Ser Val GlyIle His Ala Cys Arg Arg Ala Asn Val Cys Gln Glu 180 185 190 Thr Asn ValLeu Val Val Gly Ala Gly Pro Ile Gly Leu Val Thr Leu 195 200 205 Leu AlaAla Arg Ala Phe Gly Ala Pro Arg Ile Val Ile Ala Asp Val 210 215 220 AsnAsp Glu Arg Leu Leu Ile Ala Lys Ser Leu Gly Ala Asp Ala Val 225 230 235240 Val Lys Val Ser Thr Asn Ile Glu Asp Val Ala Glu Glu Val Ala Lys 245250 255 Ile Gln Lys Val Leu Glu Asn Gly Val Asp Val Thr Phe Asp Cys Ala260 265 270 Gly Phe Asn Lys Thr Ile Thr Thr Ala Leu Ser Ala Thr Arg ProGly 275 280 285 Gly Lys Val Cys Leu Val Gly Met Gly Gln Arg Glu Met ThrLeu Pro 290 295 300 Leu Ala Thr Arg Glu Ile Asp Val Ile Gly Ile Phe ArgTyr Gln Asn 305 310 315 320 Thr Trp Pro Leu Cys Leu Glu Phe Leu Arg SerGly Lys Ile Asp Val 325 330 335 Lys Pro Leu Ile Thr His Arg Phe Gly PheSer Gln Lys Glu Val Glu 340 345 350 Glu Ala Phe Glu Thr Ser Ala Arg GlyGly Asn Ala Ile Lys Val Met 355 360 365 Phe Asn Leu 370

What is claimed is:
 1. An isolated nucleic acid fragment encoding an aldehyde reductase comprising a member selected from the group consisting of: (a) an isolated nucleic acid fragment encoding an amino acid sequence with at least 300 contiguous amino acids and that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:2, 4, 6 and 8; (b) an isolated nucleic acid fragment that is complementary to (a).
 2. The isolated nucleic acid fragment of claim 1 wherein nucleic acid fragment is a functional RNA.
 3. The isolated nucleic acid fragment of claim 1 wherein the nucleotide sequence of the fragment comprises the sequence set forth in a member selected from the group consisting of SEQ ID NO:1, 3,5 and
 7. 4. A chimeric gene comprising the nucleic acid fragment of claim 1 operably linked to suitable regulatory sequences.
 5. A transformed host cell comprising the chimeric gene of claim
 4. 6. An aldehyde reductase polypeptide comprising the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NO:2, 4, 6 and
 6. 7. An isolated nucleic acid fragment encoding a NADPH-dependent mannose 6-phosphate reductase comprising a member selected from the group consisting of: (a) an isolated nucleic acid fragment encoding an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:10, 12 and 14, wherein the amino acid sequence encodes a functional enzyme; (b) an isolated nucleic acid fragment that is complementary to (a).
 8. The isolated nucleic acid fragment of claim 7 wherein nucleic acid fragment is a functional RNA.
 9. The isolated nucleic acid fragment of claim 7 wherein the nucleotide sequence of the fragment comprises the sequence set forth in a member selected from the group consisting of SEQ ID NO:9, 11 and
 13. 10. A chimeric gene comprising the nucleic acid fragment of claim 7 operably linked to suitable regulatory sequences.
 11. A transformed host cell comprising the chimeric gene of claim
 10. 12. A NADPH-dependent mannose 6-phosphate reductase polypeptide comprising the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NO:10, 12 and
 14. 13. An isolated nucleic acid fragment encoding a sorbitol dehydrogenase comprising a member selected from the group consisting of: (a) an isolated nucleic acid fragment encoding an amino acid sequence of at least 150 contiguous amino acids wherein the amino acid sequence is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:16, 18, 20 and 22; (b) an isolated nucleic acid fragment that is complementary to (a).
 14. The isolated nucleic acid fragment of claim 13 wherein nucleic acid fragment is a functional RNA.
 15. The isolated nucleic acid fragment of claim 13 wherein the nucleotide sequence of the fragment comprises the sequence set forth in a member selected from the group consisting of SEQ ID NO:15, 17, 19 and
 21. 16. A chimeric gene comprising the nucleic acid fragment of claim 13 operably linked to suitable regulatory sequences.
 17. A transformed host cell comprising the chimeric gene of claim
 16. 18. A sorbitol dehydrogenase polypeptide comprising the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NO:16, 18, 20 and
 22. 19. A method of altering the level of expression of a sorbitol biosynthetic enzyme in a host cell comprising: (a) transforming a host cell with the chimeric gene of any of claims 4, 10 and 16; and (b) growing the transformed host cell produced in step (a) under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of a sorbitol biosynthetic enzyme in the transformed host cell.
 20. A method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a sorbitol biosynthetic enzyme comprising: (a) probing a cDNA or genomic library with the nucleic acid fragment of any of claims 1, 7 and 13; (b) identifying a DNA clone that hybridizes with the nucleic acid fragment of any of claims 1, 7 and 13; (c) isolating the DNA clone identified in step (b); and (d) sequencing the cDNA or genomic fragment that comprises the clone isolated in step (c) wherein the sequenced nucleic acid fragment encodes all or a substantial portion of the amino acid sequence encoding a sorbitol biosynthetic enzyme.
 21. A method of obtaining a nucleic acid fragment encoding a substantial portion of an amino acid sequence encoding a sorbitol biosynthetic enzyme comprising: (a) synthesizing an oligonucleotide primer corresponding to a portion of the sequence set forth in any of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21; and (b) amplifying a cDNA insert present in a cloning vector using the oligonucleotide primer of step (a) and a primer representing sequences of the cloning vector wherein the amplified nucleic acid fragment encodes a substantial portion of an amino acid sequence encoding a sorbitol biosynthetic enzyme.
 22. The product of the method of claim
 20. 23. The product of the method of claim
 21. 